transforming-growth-factor-beta and Lymphoma--B-Cell

The process of apoptosis is used to eliminate unwanted cells from a wide variety of organisms. Various extracellular signals, often converging in common intracellular pathways, can induce apoptosis in a cell-type-specific fashion. Recent work from several laboratories has demonstrated that Rel/NF-kappa B transcription factors regulate apoptosis in many cell types. In most cells, Rel/NF-kappa B transcription factors appear to mediate survival signals that protect cells from apoptosis; however, under some circumstances, activation of these factors may also promote apoptosis.

Topics: Animals; Apoptosis; B-Lymphocytes; bcl-X Protein; CD40 Antigens; Cell Survival; Genes, Regulator; Humans; Ligands; Lymphoma, B-Cell; NF-kappa B; Oncogenes; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Transcription Factor RelB; Transcription Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Topics: Animals; Apoptosis; CD40 Ligand; Genes, myc; Immunoglobulin M; Lymphoma, B-Cell; Membrane Glycoproteins; Mice; NF-kappa B; RNA, Messenger; RNA, Neoplasm; Tosylphenylalanyl Chloromethyl Ketone; Transforming Growth Factor beta; Tumor Cells, Cultured

TGF-beta 1 and TGF-beta 2 are equipotent selective inhibitors of murine and human haemopoiesis in vitro. Primitive haemopoietic cells such as the high proliferative potential progenitor cell and the colony-forming unit (CFU)-GEMM are directly inhibited by TGF-beta whereas the more differentiated CFU-G, CFU-M and CFU-E are not. Recombinant TGF-beta 1 administered intraperitoneally or intravenously to mice selectively inhibits haemopoietic colony formation in a time- and dose-dependent manner to the same extent as seen in vitro. The progenitors are reversibly prevented from entering the cell cycle. This inhibitory action of TGF-beta functions on at least two levels: (1) down-modulation of the cell surface expression of receptors for growth stimulatory molecules and (2) interference with the intracellular signalling pathways of these molecules. In addition, expression of TGF-beta receptors is regulated during cytokine stimulation of haemopoiesis. Neoplastic B lymphocytes can proliferate by escaping from a TGF-beta-mediated autocrine inhibitory loop. Activation signals (e.g. phorbol esters) inhibit tumour cell growth by stimulating active TGF-beta production and inducing cell surface expression of TGF-beta receptors. These results indicate that TGF-beta may be useful as a bone marrow protective and/or an antitumour agent.

Topics: Animals; Antineoplastic Agents; B-Lymphocytes; Cell Cycle; Cells, Cultured; Cytokines; Down-Regulation; Drug Synergism; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoiesis; Hematopoietic Stem Cells; Humans; Immunologic Factors; Leukemia, Promyelocytic, Acute; Lymphoma, B-Cell; Mice; Neoplastic Stem Cells; Receptors, Cell Surface; Receptors, Transforming Growth Factor beta; Recombinant Proteins; Transforming Growth Factor beta; Tumor Cells, Cultured

Chimeric antigen receptor (CAR)-T cells are effective in the treatment of hematologic malignancies but have shown limited efficacy against solid tumors. Here, we demonstrated an approach to inhibit recurrence of B cell lymphoma by co-expressing both a human anti-CD19-specific single-chain variable fragment (scFv) CAR (CD19 CAR) and a TGF-β/IL-7 chimeric switch receptor (tTRII-I7R) in T cells (CD19 CAR-tTRII-I7R-T cells). The tTRII-I7R was designed to convert immunosuppressive TGF-β signaling into immune-activating IL-7 signaling. The effect of TGF-β on CD19 CAR-tTRII-I7R-T cells was assessed by western blotting. Target-specific killing by CD19 CAR-tTRII-I7R-T cells was evaluated by Eu-TDA assay. Daudi tumor-bearing NSG (NOD/SCID/IL2Rγ

Topics: Animals; Antigens, CD19; Cells, Cultured; Female; Humans; Immunotherapy, Adoptive; Interleukin-7; K562 Cells; Lymphoma, B-Cell; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Recurrence, Local; Receptors, Chimeric Antigen; Signal Transduction; Single-Chain Antibodies; Transforming Growth Factor beta; Treatment Outcome; Xenograft Model Antitumor Assays

Regulatory T cells (Tregs) play essential roles in maintaining immunological self-tolerance and preventing autoimmunity. The adoptive transfer of antigen-specific Tregs has been expected to be a potent therapeutic method for autoimmune diseases, severe allergy, and rejection in organ transplantation. However, effective Treg therapy has not yet been established because of the difficulty in preparing a limited number of antigen-specific Tregs. Chimeric antigen receptor (CAR) T cells have been shown to be a powerful therapeutic method for treating B cell lymphomas, but application of CAR to Treg-mediated therapy has not yet been established. Here, we generated CD19-targeted CAR (CD19-CAR) Tregs from human PBMCs (hPBMCs) and optimized the fraction of the Treg source as CD4+CD25+CD127loCD45RA+CD45RO-. CD19-CAR Tregs could be expanded in vitro while maintaining Treg properties, including high expression of the latent form of TGF-β. CD19-CAR Tregs suppressed IgG antibody production and differentiation of B cells via a TGF-β-dependent mechanism. Unlike conventional CD19-CAR CD8+ T cells, CD19-CAR Tregs suppressed antibody production in immunodeficient mice that were reconstituted with hPBMCs, reducing the risk of graft-versus-host disease. Therefore, the adoptive transfer of CD19-CAR Tregs may provide a novel therapeutic method for treating autoantibody-mediated autoimmune diseases.

Topics: Adoptive Transfer; Animals; Antigens, CD19; Autoimmunity; B-Lymphocytes; Epitopes; Graft vs Host Disease; Humans; Immune Tolerance; Immunoglobulin G; Lymphoma, B-Cell; Mice; Receptors, Chimeric Antigen; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Chimeric antigen receptor (CAR)-expressing T cells targeting surface-bound tumor antigens have yielded promising clinical outcomes, with two CD19 CAR-T cell therapies recently receiving FDA approval for the treatment of B-cell malignancies. The adoption of CARs for the recognition of soluble ligands, a distinct class of biomarkers in physiology and disease, could considerably broaden the utility of CARs in disease treatment. In this study, we demonstrate that CAR-T cells can be engineered to respond robustly to diverse soluble ligands, including the CD19 ectodomain, GFP variants, and transforming growth factor beta (TGF-β). We additionally show that CAR signaling in response to soluble ligands relies on ligand-mediated CAR dimerization and that CAR responsiveness to soluble ligands can be fine-tuned by adjusting the mechanical coupling between the CAR's ligand-binding and signaling domains. Our results support a role for mechanotransduction in CAR signaling and demonstrate an approach for systematically engineering immune-cell responses to soluble, extracellular ligands.

Topics: Antigens, CD19; Biomarkers; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cytokines; Green Fluorescent Proteins; Humans; Immunosuppressive Agents; Ligands; Lymphoma, B-Cell; Protein Domains; Protein Engineering; Protein Multimerization; Receptors, Chimeric Antigen; T-Lymphocytes; Transforming Growth Factor beta

Transforming growth factor beta (TGF-β) has an important role in mediating T-cell suppression in B-cell non-Hodgkin lymphoma (NHL). However, the underlying mechanism responsible for TGF-β-mediated inhibition of effector memory T (Tm) cells is largely unknown. As reported here, we show that exhaustion is a major mechanism by which TGF-β inhibits Tm cells, and TGF-β mediated exhaustion is associated with upregulation of CD70. We found that TGF-β upregulates CD70 expression on effector Tm cells while it preferentially induces Foxp3 expression in naive T cells. CD70 induction by TGF-β is Smad3-dependent and involves IL-2/Stat5 signaling. CD70+ T cells account for TGF-β-induced exhaustion of effector Tm cells. Both TGF-β-induced and preexisting intratumoral CD70+ effector Tm cells from B-cell NHL have an exhausted phenotype and express higher levels of PD-1 and TIM-3 compared with CD70- T cells. Signaling transduction, proliferation and cytokine production are profoundly decreased in these cells, and they are highly susceptible to apoptosis. Clinically, intratumoral CD70-expressing T cells are prevalent in follicular B-cell lymphoma (FL) biopsy specimens, and increased numbers of intratumoral CD70+ T cells correlate with an inferior patient outcome. These findings confirm TGF-β-mediated effector Tm cell exhaustion as an important mechanism of immune suppression in B-cell NHL.

Topics: Apoptosis; CD27 Ligand; Gene Expression Regulation, Neoplastic; Hepatitis A Virus Cellular Receptor 2; Humans; Immunologic Memory; Interleukin-2; Lymphoma, B-Cell; Membrane Proteins; Programmed Cell Death 1 Receptor; Signal Transduction; STAT5 Transcription Factor; T-Lymphocytes; Transforming Growth Factor beta; Tumor Necrosis Factor Receptor Superfamily, Member 7

FOX genes encode transcription factors which regulate basic developmental processes during embryogenesis and in the adult. Several FOX genes show deregulated expression in particular malignancies, representing oncogenes or tumor suppressors. Here, we screened six Hodgkin lymphoma (HL) cell lines for FOX gene activity by comparative microarray profiling, revealing overexpression of FOXC1 and FOXD1, and reduced transcription of FOXN3, FOXO1, and FOXP1. In silico expression analyses of these FOX gene candidates in HL patient samples supported the cell line data. Chromosomal analyses demonstrated an amplification of the FOXC1 locus at 6p25 and a gain of the FOXR2 locus at Xp11, indicting genomic aberrations for their upregulation. Comparative expression profiling and ensuing stimulation experiments revealed implementation of the TGFβ- and WNT-signaling pathways in deregulation of FOXD1 and FOXN3. Functional analysis of FOXP1 implicated miR9 and miR34a as upstream regulators and PAX5, TCF3, and RAG2 as downstream targets. A similar exercise for FOXC1 revealed repression of MSX1 and activation of IPO7, both mediating inhibition of the B-cell specific homeobox gene ZHX2. Taken together, our data show that aberrantly expressed FOX genes and their downstream targets are involved in the pathogenesis of HL via deregulation of B-cell differentiation and may represent useful diagnostic markers and/or therapeutic targets.

Topics: Burkitt Lymphoma; Cell Line, Tumor; Chromosomes, Human; Forkhead Transcription Factors; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Genetic Loci; Hodgkin Disease; Homeodomain Proteins; Humans; Karyopherins; Lymphoma, B-Cell; Lymphoma, Follicular; MicroRNAs; MSX1 Transcription Factor; Receptors, Cytoplasmic and Nuclear; Transcription Factors; Transforming Growth Factor beta; Wnt Signaling Pathway

Transforming growth factor-β (TGF-β) exhibits growth inhibitory effects on various types of tumor cells, including B-cell lymphoma cells. In the present study, the role of TGF-β in the survival of Epstein-Barr virus-negative B-cell lymphoma Ramos cells was investigated. As TGF-β-induced apoptosis of Ramos cells in vitro and in vivo, we attempted to identify novel target gene(s) responsible for their survival. Oligonucleotide microarray analysis and chromatin immunoprecipitation revealed that Smad proteins directly regulated the transcription of membrane-spanning 4-domains, subfamily A, member 1 (MS4A1), also known as CD20, in Ramos cells upon TGF-β stimulation. In addition, immunohistochemical analysis using clinical samples from B-cell lymphoma patients showed an inverse correlation between the expression of MS4A1/CD20 and phosphorylation of Smad3. Although knockdown of MS4A1/CD20 in Ramos cells resulted in an increase of apoptotic cells, Ramos cells stably expressing MS4A1/CD20 were resistant to TGF-β-induced apoptosis. This suggests that MS4A1/CD20 is responsible for TGF-β-induced apoptosis of B-cell lymphoma cells. Moreover, downregulation of MS4A1/CD20 by TGF-β attenuated the effects of the monoclonal anti-MS4A1/CD20 antibody, rituximab, on Ramos cells. Our findings suggest that the sensitivity of B-cell lymphoma cells to rituximab may be affected by TGF-β signaling.

Topics: Animals; Antibodies, Monoclonal, Murine-Derived; Antigens, CD20; Antineoplastic Agents; Apoptosis; Burkitt Lymphoma; Cell Line, Tumor; Humans; Lymphoma, B-Cell; Male; Mice; Mice, Inbred BALB C; Rituximab; Signal Transduction; Transforming Growth Factor beta

c-Myc transformed human Burkitt's lymphoma (BL) cells are highly sensitive to TGF-β-induced apoptosis. Previously we demonstrated that TGF-β-mediated cell death in BL cells is regulated via the mitochondrial intrinsic apoptosis pathway, which is dependent on the activation of BAX and/or BAK. TGF-β directly induces transcription of the BH3-only protein BIK and represses expression of the pro-survival factor BCL-X(L) but has no effect on the direct BAX/BAK "activators" BIM or BID (tBID). Here we show that TGF-β induces the BH3-only activator PUMA to aid induction of the intrinsic cell death pathway. TGF-β also induced PUMA in normal germinal center CD77-positive centroblasts isolated from human tonsil tissue. PUMA was a direct TGF-β target gene in B-cells, and we identify a putative Smad-binding region within the human PUMA promoter that recruits Smad3 and Smad4 in cells in response to TGF-β signaling. Constitutive activity of the isolated Smad-binding region in luciferase reporter assays was dependent on Smad consensus sequences and was partially dependent on endogenous TGF-β signaling and Smad4. Knockdown of PUMA in BL cells using lentiviral shRNA resulted in slower kinetics of the TGF-β-mediated apoptotic response. Analysis of Eμ-Myc cell lines demonstrated that c-myc-driven murine lymphomas are also sensitive to TGF-β-mediated apoptosis. Moreover, Puma(-/-) Eμ-Myc lines demonstrated significantly delayed kinetics of the apoptotic response when compared with wild type lymphomas. TGF-β therefore induces a polygenic response in Myc-driven lymphomas involving transcription of PUMA, which is necessary for the rapid induction of cell death.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Cell Line; Cell Survival; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Kinetics; Lymphoma; Lymphoma, B-Cell; Mice; Promoter Regions, Genetic; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myc; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta; Tumor Suppressor Protein p53

Notch-signalling has been implicated as a pathogenetic factor and a therapeutical target in T-cell leukaemias and in some lymphomas of B-cell origin. Our aim was to investigate the role of Notch-signalling in apoptosis regulation in human non-Hodgkin B-cell lymphoma (B-NHL) cell lines and in primary chronic lymhocytic leukaemia (CLL) cells using Delta-like 4 (Dll4) ligand mediated Notch activation and gamma-secretase inhibitor (GSI) mediated Notch inhibition in vitro. The potential cross-talk of Notch with the transforming growth factor-beta (TGFb) pathway in apoptosis induction was also explored, and the effect of GSI on drug-induced apoptosis was assessed. Modulation of Notch-signalling by itself did not change the rate of apoptosis in B-NHL cell lines and in CLL cells. TGFb-induced apoptosis was decreased - but not completely abolished - by GSI in TGFb-sensitive cell lines, but resistance to the apoptotic effects of TGFb were not reversed by Notch activation or inhibition. Drug-induced apoptosis was not modified by GSI. We identified Hairy/Enhancer of Split (HES)-1 as a TGFb target gene in selected - TGFb-sensitive - B-NHL cell lines. TGFb-induced HES-1 was only partially Notch-dependent in later phases. Apoptosis regulation by TGFb and GSI was not dependent on the transcriptional regulation of c-myc. In conclusion, our data does not support a unifying role of Notch in regulating apoptosis in B-NHL, but warns that gamma-secretase inhibitors may actually counteract apoptosis in some cases.

Topics: Adaptor Proteins, Signal Transducing; Amyloid Precursor Protein Secretases; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Calcium-Binding Proteins; Cell Line, Tumor; Dipeptides; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Intercellular Signaling Peptides and Proteins; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Receptors, Notch; Repressor Proteins; Signal Transduction; Transcription Factor HES-1; Transforming Growth Factor beta

Cytokines of the transforming growth factor β (TGF-β) superfamily exert effects on proliferation, apoptosis and differentiation in various cell types. Cancer cells frequently acquire resistance to the anti-proliferative signals of TGF-β, which can be due to mutations in proteins of the signalling cascade. We compared the TGF-β-related signalling properties in B-cell lymphoma cell lines that were sensitive or resistant to TGF-β-induced anti-proliferative effects.. TGF-β sensitive cell lines expressed higher cell surface levels of the activin receptor-like kinase 5 (Alk-5), a TGF-β receptor type 1. The expression levels of the other TGF-β and bone morphogenetic protein receptors were comparable in the different cell lines. TGF-β-induced phosphorylation of Smad2 was similar in TGF-β sensitive and resistant cell lines. In contrast, activation of Smad1/5 was restricted to cells that were sensitive to growth inhibition by TGF-β. Moreover, with activin A we detected limited anti-proliferative effects, strong phosphorylation of Smad2, but no Smad1/5 phosphorylation. Up-regulation of the TGF-β target genes Id1 and Pai-1 was identified in the TGF-β sensitive cell lines. Constitutive phosphorylation of MAPK p38 was restricted to the TGF-β sensitive cell lines. Inhibition of p38 MAPK led to reduced sensitivity to TGF-β.. We suggest that phosphorylation of Smad1/5 is important for the anti-proliferative effects of TGF-β in B-cell lymphoma. Alk-5 was highly expressed in the sensitive cell lines, and might be important for signalling through Smad1/5. Our results indicate a role for p38 MAPK in the regulation of TGF-β-induced anti-proliferative effects.

Topics: B-Lymphocytes; Bone Morphogenetic Protein Receptors; Cell Line, Tumor; Cell Proliferation; Enzyme Activation; Growth Inhibitors; Humans; Lymphoma, B-Cell; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad1 Protein; Smad5 Protein; Transforming Growth Factor beta

Using biopsy specimens from patients with B-cell non-Hodgkin's lymphoma, we observed a significantly low frequency of T(H)17 cells, including several samples with no detectable amount of interleukin (IL)-17-producing cells present in the tumor microenvironment. We found that, in the absence of lymphoma B cells, treatment with IL-1beta/IL-6 or lipopolysaccharide (LPS) enhanced IL-17 expression in CD4(+) T cells and this enhancement was attenuated when CD4(+) T cells were cocultured with lymphoma B cells. Blockade of CD27-CD70 or CD28-CD80/86 interactions by anti-CD70 or anti-CD80/86 antibodies restored LPS-mediated induction of IL-17 expression in CD4(+) T cells cocultured with lymphoma B cells. Because a subset of lymphoma B cells express IL-2 and given that IL-2 signaling is critically important in the development of regulatory T (T(reg)) cells, we tested the role of IL-2 signaling in T(H)17 cell development. We found that treatment with anti-IL-2 antibody to interrupt IL-2 signaling significantly inhibited Foxp3 expression in CD4(+) T cells. In contrast, interruption of IL-2 signaling up-regulated IL-17 expression in CD4(+) T cells and restored lymphoma-mediated down-regulation of IL-17-producing cells. Furthermore, the reversal of T(reg) cell activity by LPS or CpG-A resulted in an enhancement of IL-17-producing cells. Taken together, our study indicated that lymphoma B cells play an important role in skewing the balance between T(reg) and T(H)17 cells resulting in the establishment of a profoundly inhibitory tumor microenvironment.

Topics: B-Lymphocytes; Biopsy; CD4-Positive T-Lymphocytes; Cytokines; Flow Cytometry; Forkhead Transcription Factors; Humans; Interleukin-17; Interleukin-2; Interleukin-23; Lymphoma, B-Cell; Lymphoma, Non-Hodgkin; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) controls a wide spectrum of cellular processes. Deregulation of TGF-beta signaling contributes to the pathogenesis of many diseases including cancer and autoimmune diseases. TGF-beta signaling is generally mediated through intracellular signal transducers and transcription factors called Smads. Herein, we have identified the oncoprotein BCL6 as a transcriptional corepressor of tumor suppressor Smad4. BCL6 physically interacts with Smad3 and Smad4, disrupts the Smad-p300 interaction, and represses the transcriptional activity of Smad4. In accordance, B-cell lymphoma cells with a high expression level of BCL6 were found to be refractory to TGF-beta antiproliferative response, whereas knockdown of BCL6 expression in B-cell lymphoma cells partially restores the TGF-beta responses. This study provides strong evidence that overexpression of BCL6 contributes to TGF-beta resistance in B-cell lymphoma.

Topics: Animals; Burkitt Lymphoma; Cell Growth Processes; Cell Line, Tumor; Chromatin; DNA-Binding Proteins; E1A-Associated p300 Protein; Humans; Lymphoma, B-Cell; Proto-Oncogene Proteins c-bcl-6; Rats; RNA, Small Interfering; Signal Transduction; Smad4 Protein; Transcription, Genetic; Transforming Growth Factor beta

Membrane-bound and soluble human leucocyte antigen-G (sHLA-G) molecules display immunotolerant properties favouring tumour cell escape from immune surveillance. sHLA-G molecules have been detected in several tumour pathologies; this study aimed to evaluate sHLA-G expression in lymphoproliferative disorders. sHLA-G plasma level was significantly increased in 110 of 178 newly diagnosed lymphoid proliferations cases i.e. 59% of chronic lymphocytic leukaemia, 65% of B non-Hodgkin lymphomas (NHL) and 58% of T-NHL. To assess the mechanisms involved in this secretion, the differential effect of cytokines was tested in in vitro cultures of NHL cells. A significant induction of sHLA-G level was shown in T-NHL in contrast with B-NHL and normal equivalent cells, after cytokine stimulation with (i) interferongamma (IFNgamma), interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor, (ii) IL-10 and (iii) transforming growth factor beta. An impact of microenvironment on sHLA-G expression was found in B-NHL as shown by the in vitro effect of addition of normal monocytes. Finally, a functional effect of sHLA-G molecules purified from pathologic plasma was demonstrated by their strong capacity to inhibit T-cell proliferation at concentrations currently observed during these disorders. These results suggest that functional sHLA-G molecules are upregulated in lymphoproliferative disorders which can support their potential immunomodulatory role during this pathology.

Topics: B-Lymphocytes; Cell Division; Granulocyte-Macrophage Colony-Stimulating Factor; Histocompatibility Antigens Class I; HLA Antigens; HLA-G Antigens; Humans; Interferon-gamma; Interleukins; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Lymphoma, T-Cell; Lymphoproliferative Disorders; Monocytes; Prospective Studies; T-Lymphocytes; Transforming Growth Factor beta; Tumor Cells, Cultured

Non-Hodgkin's lymphomas (NHL) constitute a heterogenous group of mainly B-cell lymphoproliferative diseases with different patterns of clinical behaviour. Biological mechanisms leading to development of NHL are not clearly understood. Transforming growth factor-beta1 (TGF-beta1) influences B cell growth and development. The present study aimed to determine whether there is an association between the polymorphic features located within the TGF-beta1 gene in NHL patients and progression of the disease. Two single nucleotide polymorphisms at positions 869 T/C (Leu10Pro) and 915 G/C (Arg25Pro) in the precursor region of the TGF-beta1 gene were determined in 55 NHL patients and 50 healthy individuals by PCR-SSP technique using commercial primers. In univariate analysis the presence of TGF-beta1 high producer genotypes (T/T G/G or T/C G/G) was found to significantly associate with an increased number of extranodal sites (11/30 vs 3/25, p=0.035 for two or more extranodal sites in patients having or lacking the TGF-beta1 high producer genotype, respectively). TGF-beta1 high producer genotype together with other clinical and biological factors (patient sex and age, stage and aggressiveness of the disease, presence of B symptoms, serum LDH level) were subjected to multivariate logistic regression analyses for the number of extranodal sites. Multivariate analysis confirmed the role of TGF-beta1 high producer genotype as a risk factor of NHL manifestation in two or more extranodal sites (OR=7.217, p=0.043) in addition to histological aggressiveness of the disease (OR=4.302, p=0.057). TGF-beta1 gene polymorphisms were found to associate with the course of the disease in NHL patients. TGF-beta1 high producer genotype appeared as an independent risk factor of extranodal manifestation of the disease.

Topics: Alleles; Disease Progression; Female; Genotype; Humans; Lymphoma, B-Cell; Lymphoma, Non-Hodgkin; Male; Middle Aged; Polymorphism, Genetic; Risk Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1

TGFbeta1 has a profound effect on the growth of various mammalian cell types, including B lymphocytes. The inhibitory action of TGFbeta1 is mediated by deactivation of the cell cycle machinery. Several feedback-sensitive pathways determine whether the cells are stopped in G1 phase or allowed to leave G1 phase and enter S phase. Cell cycle-associated molecules, e.g. cyclin-dependent kinase inhibitors (CKIs), may become targets for the inhibitory signaling pathways induced by TGFbeta1.. Our experimental DoHH2 cell line model was derived from a patient with malignant non-Hodgkin's lymphoma of follicular origin. The effect of TGFbeta1 on cell cycle progression was studied by flow cytometry. We examined the effect of TGFbeta1 on the expression of p21WAF1 by immunoblotting and RT-PCR. The binding activity of transcription factors to the p21 gene promoter was determined by gel mobility shift assay (GMSA).. Our results showed that TGFbeta1 treatment increased the number of cells arrested in G0/G1 phase compared with untreated control cells. Moreover, we found that p21WAF1 expression was significantly up-regulated on the protein level after TGFbeta1 treatment. Similarly to the protein level, the expression of p21 mRNA was increased in TGFbeta1-treated cells. We further examined the binding activity of the Sp family of transcription factors to examine their role in p21WAF1 up-regulation.. The results indicated that p21WAF1 over-expression in TGFbeta1-arrested malignant B cells is mediated by binding of Sp1/Sp3 transcription factors to the (-92/-71), (-77/-58), and (-65/-45) elements of the promoter region of the p21 gene.

Topics: B-Lymphocytes; Base Sequence; Blotting, Western; Cell Cycle; Cell Line; Cyclin-Dependent Kinase Inhibitor p21; DNA Primers; Electrophoretic Mobility Shift Assay; Humans; Lymphoma, B-Cell; Reverse Transcriptase Polymerase Chain Reaction; Sp1 Transcription Factor; Sp3 Transcription Factor; Transforming Growth Factor beta; Up-Regulation

The early B-cell factor (EBF)-associated zinc-finger protein (EBFAZ) binds to and negatively regulates EBF, a basic helix-loop-helix transcription factor required for B-cell lineage commitment and development of the olfactory epithelium. It also binds to SMA- and MAD-related protein 1 (SMAD1) and SMAD4 in response to bone morphogenic protein 2 (BMP2) signaling. It is highly related to ecotropic viral integration site 3 (EVI3), a protein that, like EBFAZ, contains 30 Krüppel-like zinc-finger repeats. In previous studies, we showed that Evi3 is a frequent target of retroviral integration in AKXD27 B-cell lymphomas. Here, we show that EBFAZ is also a frequent target. Integrations at Ebfaz and Evi3 are mutually exclusive, suggesting that they function in the same tumor pathway. Lymphomas with integrations at Ebfaz or Evi3 express the pre-B-cell-specific marker immunoglobulin lambda chain 5, and contain immunoglobulin heavy-chain rearrangements, suggesting that they are blocked at an early B-cell stage. Unlike Evi3, which is expressed at low levels in normal B cells, or Ebfaz, which is not expressed in B cells, both genes are highly expressed following viral integration. Collectively, our results suggest that ectopic expression of Ebfaz can substitute for the upregulated expression of Evi3 in B-cell disease and highlight the importance of this gene family in hematopoietic cancer.

Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Southern; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; DNA-Binding Proteins; DNA, Complementary; Exons; Immunoglobulin Heavy Chains; Lymphoma; Lymphoma, B-Cell; Mice; Models, Genetic; Molecular Sequence Data; Protein Binding; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Smad Proteins; Smad1 Protein; Smad4 Protein; Tissue Distribution; Trans-Activators; Transcription Factors; Transforming Growth Factor beta; Up-Regulation; Virus Integration

Pulmonary fibrosis is a severe complication associated with bis-chloronitrosourea (BCNU) therapy. However, the pathogenetic mechanism has never been well investigated. We report here a 26-year-old female with diffuse large B-cell lymphoma who died of severe pulmonary fibrosis 81 days after the administration of high-dose BCNU (600 mg/m2). Thoracoscopic wedge resection of left upper lung performed 10 days before patient's death showed severe pulmonary fibrosis with prominent hyperplasia of alveolar macrophages and type II pneumocytes. We further used immunohistochemistry (IHC) to examine the relative role of platelet-derived growth factor-B (PDGF-B), insulin-like growth factor I (IGF-I), transforming growth factor-beta1 (TGF-beta1) and cyclooxygenase-2 (COX-2) in the pathogenesis of BCNU-related pulmonary fibrosis. Strong expressions of PDGF-B and IGF-1 on alveolar macrophages and type II pneumocytes were clearly demonstrated, but in contrast, the expressions of TGF-beta1 and COX-2 were almost undetectable. In conclusion, pulmonary fibrosis can develop early and progress rapidly after the administration of high-dose BCNU. The markedly increased expression of fibrogenic factors PDGF-B and IGF-1 on hyperplastic alveolar macrophages and hyperplastic type II pneumocytes may play an important role in the fibrogenesis of this disease. These novel findings may offer specific therapeutic targets in the treatment of BCNU-associated pulmonary fibrosis.

Topics: Adult; Antineoplastic Agents, Alkylating; Carmustine; Cyclooxygenase 2; Fatal Outcome; Female; Humans; Insulin-Like Growth Factor I; Isoenzymes; Lung; Lymphoma, B-Cell; Lymphoma, Large B-Cell, Diffuse; Membrane Proteins; Prostaglandin-Endoperoxide Synthases; Proto-Oncogene Proteins c-sis; Pulmonary Fibrosis; Transforming Growth Factor beta; Transforming Growth Factor beta1

We have previously reported an overexpression of Smad1 in follicular lymphoma (FL) cells, which are characterized by the t(14;18) bcl2/IgH translocation. Smad1 is commonly involved in bone morphogenetic protein but not in tumor-transforming growth factor beta (TGFbeta) signaling pathways. This study focuses on Smad1 signaling pathway in non-Hodgkin lymphoma cells including follicular or large-cell lymphoma cells. Our results support the notion that phosphorylation of Smad1 is mediated by TGFbeta present in the microenvironment and occurs in FL in vivo. Using an in vitro coculture system mimicking interactions between stroma cells and FL cells, we found that both the cell partners release TGFbeta at a sufficient concentration to activate Smad pathways in the malignant cells. This Smad1 activation involves TGFbetaRII but not ALK-1 receptors, and does not compete with the Smad2 pathway. Moreover, proliferation assays performed on lymphoma cells expressing wild-type or mutated Smad1, or in which endogenous Smad1 level was decreased by gene silencing, strongly supported that overexpression and activation of Smad1 modifies the biological response of lymphoma B cells to TGFbeta family members. This work opens new insights into aberrant Smad pathways and their pathophysiological role in FL and in other non-Hodgkin lymphomas.

Topics: Activin Receptors, Type I; Activin Receptors, Type II; B-Lymphocytes; Cell Proliferation; DNA-Binding Proteins; Gene Silencing; Humans; Lymphoma, B-Cell; Lymphoma, Follicular; Lymphoma, Large B-Cell, Diffuse; Mutation; Palatine Tonsil; Phosphorylation; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Smad Proteins; Smad1 Protein; Smad2 Protein; Stromal Cells; Trans-Activators; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor beta1 (TGFbeta1) induces growth arrest in many cell types, including B lymphocytes. The inhibitory action of TGFbeta1 is mediated by the deactivation of kinase complexes. The cell-cycle engine is tightly controlled by cyclin-dependent kinase (cdk) inhibitors, which mediate extracellular negative signals, resulting in cell-cycle arrest at different G1 points.. Our experimental DoHH2 cell line model was derived from a patient with malignant non-Hodgkin's lymphoma (NHL) of follicular origin. We examined the effect of TGFbeta1 on the expression and phosphorylation of key cell-cycle regulators by immunoprecipitation and immunoblotting.. After 48 hours of TGFbeta1 (10 ng/ml) treatment, a significantly increased number of DoHH2 cells was retained in G0/G1 phase. Our results showed the inhibitory action was associated with hypophosphorylation of pRb on serine 795 (S795) and threonine 373 (T373). We examined the composition of the cdk complexes and the level of cdk inhibitors to explain the inhibitory action of TGFbeta1 on cdk activity. Western blotting showed that the total level of the kinase inhibitor p21 (WAF1) increased after TGFbetal treatment. Our results indicate that a notably high level of p21(WAF1) was bound to cdk4/6 due to the treatment and that the binding of p21(WAF1) was associated with cyclin D-cdk4/6 complex decomposition.. Our investigation of the effect of TGFbetal on cell-cycle progression of a non-Hodgkin's lymphoma cell line of follicular lymphoma subtype showed that the TGFbeta1-induced growth arrest of malignant B cells was associated with the activation of CIP/KIP family members of cdk inhibitors.

Topics: Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Humans; Lymphoma, B-Cell; Lymphoma, Follicular; Male; Middle Aged; Phosphorylation; Transforming Growth Factor beta; Transforming Growth Factor beta1

A novel cell line, designated OHK, was established from ascites of a 59-year-old Japanese woman with diffuse large B-cell lymphoma showing a peculiar serosal tropism, as seen in primary effusion lymphomas (PEL). OHK exhibited a large pleomorphic morphology with irregular nuclei and distinct nucleoli, and included immunoblastic and Reed-Sternberg-like giant cells. On ultrastructural examination, rich intermediate filaments, and well-developed Golgi apparati and rough endoplasmic reticulum, were seen. Immunophenotypically, OHK lacked T and B cell-associated antigens, and had CD10, CD30, CD33 and CD138 antigens. Although OHK cells did not express immunoglobulin (Ig) protein, Southern blot analysis demonstrated clonal rearrangements of Ig heavy and light chain genes. These observations suggest that OHK cells are derived from preterminally differentiated B cells, and that they have features of PEL. Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus were not detected. OHK displayed hyperploid karyotypes with multiple structural abnormalities, and produced some cytokines such as macrophage-colony-stimulating factor (M-CSF), granulocyte-CSF, interleukin 6 and transforming growth factor beta 1. In particular, vascular endothelial growth factor (VEGF), whose stimulation of vascular permeability is thought to be critical to the pathogenesis of PEL, was also produced in large quantities. These results indicate that OHK may be a useful tool for the investigation of PEL.

Topics: Ascitic Fluid; Chromosome Aberrations; Endothelial Growth Factors; Female; Gene Rearrangement, B-Lymphocyte, Heavy Chain; Gene Rearrangement, B-Lymphocyte, Light Chain; Granulocyte Colony-Stimulating Factor; Humans; Immunophenotyping; Interleukin-6; Lymphokines; Lymphoma, B-Cell; Macrophage Colony-Stimulating Factor; Middle Aged; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Transforming growth factor (TGF)-beta1 is well established as a critical IgA isotype switching factor and Smad molecules have been reported to act as transducers and transcriptional factors in the expression of TGF-beta1-targeted genes. We examined the involvement of Smad proteins in TGF-beta1-induced IgA expression. First, we found that TGF-beta1 significantly increases endogenous germ-line (GL) alpha transcripts by LPS-stimulated CH12.LX.4933 (mu(+)) B lymphoma cells. To investigate its signaling mechanisms, the lymphoma cell line was transfected with pFL3 that contains the TGF-beta-responsive element of the GLalpha promoter, and stimulated with TGF-beta1. Similar to endogenous GLalpha transcripts, TGF-beta1 induces GLalpha promoter activity and overexpression of Smad3 markedly enhances the promoter activity. This activity is further augmented by cotransfected Smad4. On the other hand, Smad7 substantially abrogates the synergistic effect of Smad3/4 onGLalpha promoter activity. In addition, overexpression of Smad3/4 enhances TGF-beta1-induced endogenous GLalpha transcripts in normal spleen B cells. Finally, in the presence of TGF-beta1, overexpression of Smad3/4 selectively increases both surface IgA expression and IgA production. The results from the present study indicate that Smad3, Smad4, and Smad7, at least in part, serve as mediators linking TGF-beta1 to transcriptional regulation of IgA switching related gene and regulation of IgA class switching.

Topics: Animals; B-Lymphocytes; Cells, Cultured; DNA-Binding Proteins; Gene Expression Regulation; Humans; Immunoglobulin A; Lymphoma, B-Cell; Mice; Mice, Inbred BALB C; Recombinant Fusion Proteins; Smad3 Protein; Smad4 Protein; Spleen; Trans-Activators; Transcription, Genetic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Cells, Cultured

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that has a principal role in growth control through both its cytostatic effect on many different epithelial cell types and its ability to induce programmed cell death in a variety of other cell types. Here we have used a screen for proteins that interact physically with the cytoplasmic domain of the type II TGF-beta receptor to isolate the gene encoding Daxx - a protein associated with the Fas receptor that mediates activation of Jun amino-terminal kinase (JNK) and programmed cell death induced by Fas. The carboxy-terminal portion of Daxx functions as a dominant-negative inhibitor of TGF-beta-induced apoptosis in B-cell lymphomas, and antisense oligonucleotides to Daxx inhibit TGF-beta-induced apoptosis in mouse hepatocytes. Furthermore, Daxx is involved in mediating JNK activation by TGF-beta. Our findings associate Daxx directly with the TGF-beta apoptotic-signalling pathway, and make a biochemical connection between the receptors for TGF-beta and the apoptotic machinery.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Carrier Proteins; Cell Compartmentation; Cell Division; Co-Repressor Proteins; COS Cells; fas Receptor; Hepatocytes; Humans; Intracellular Signaling Peptides and Proteins; Lymphoma, B-Cell; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinases; Molecular Chaperones; Nuclear Proteins; Oligonucleotides, Antisense; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured; Two-Hybrid System Techniques; Yeasts

Using a lipopolysaccharide (LPS)-responsive murine B-cell line, CH12. LX, we assessed the possible role of CD14 in LPS-induced activation of B cells. Flow cytometric analysis indicated that CH12.LX cells expressed the CD14 molecule with a lower intensity than did the macrophage cell line J774.1. A reverse transcription-polymerase chain reaction and Northern blot analysis revealed low, but significant, levels of CD14 mRNA in CH12.LX cells, whose cDNA was identical to that of the mouse macrophage CD14 gene. After stimulation with LPS, CH12.LX cells proliferated, accompanied by up-regulations of CD14, transforming growth factor (TGF)-beta and interleukin (IL)-6 mRNA, and increased IgM and IgA secretion. In the absence of serum or with the addition of anti-CD14 monoclonal antibodies, however, LPS-stimulation induced neither the up-regulation of CD14 and TGF-beta mRNA nor an increase in IgA secretion. These findings indicate that CD14 expression is not restricted to myeloid cells, but is involved in some cellular activation events of murine B cells elicited by LPS. Furthermore, a CD14-independent pathway may also exist in the LPS-induced activation of B cells that leads to proliferation, IL-6 production and the enhancement of IgM (but not IgA) secretion.

Topics: Animals; B-Lymphocytes; Blotting, Northern; Cell Division; Cell Line; Cell Membrane; Escherichia coli; Flow Cytometry; Humans; Immunoglobulin A; Immunoglobulin M; Interleukin-6; Lipopolysaccharide Receptors; Lipopolysaccharides; Lymphocyte Activation; Lymphoma, B-Cell; Macrophages; Mice; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Up-Regulation

Transforming growth factor-beta (TGF-beta) is a potent inducer of apoptosis in B-lymphocytes and is essential for immune regulation and maintenance of self-tolerance. Here we show that concomitant signaling through CD40 sustains proliferation and rescues the premature B cell line WEHI 231 from both TGF-beta-induced and anti-IgM-induced apoptosis. The anti-apoptotic effect of CD40 is associated with the transcriptional activation of the inhibitory Smad7 protein. The transactivation of Smad7 by CD40 is NFkappaB-dependent in that pharmacological inhibitors of this pathway, N-tosyl-l-phenylalanine chloromethyl ketone and pyrrolidine dithiocarbamate, abrogate CD40-induced Smad7 expression. Ectopic overexpression of Smad7 inhibited Smad2 activation, TGF-beta-mediated growth inhibition, and apoptosis in WEHI 231 cells. Consistent with this result, dominant negative interference with Smad2 and Smad3 function also inhibited TGF-beta-induced apoptosis. The inhibitory effects of Smad7 overexpression were specific to TGF-beta-induced apoptosis and were without effect on anti-IgM-induced cell death. These results suggest a mechanism of suppression of TGF-beta-induced apoptosis by CD40, mediated through activation of NF-kappaB and, consequently, induction of Smad7 expression.

Topics: Antigens, CD; Apoptosis; B-Lymphocytes; CD40 Antigens; Cell Division; Cell Line; Cell Nucleus; DNA-Binding Proteins; Gene Expression Regulation; Humans; Lymphoma, B-Cell; Phosphorylation; Signal Transduction; Smad2 Protein; Smad7 Protein; Trans-Activators; Transfection; Transforming Growth Factor beta; Tumor Cells, Cultured

Transforming growth factor beta1 (TGFbeta1) induces growth arrest in many cell types, including B lymphocytes. We examined the effect of TGF on cell cycle progression of a non-Hodgkin lymphoma cell line of follicular lymphoma subtype (FL). After 48 h of TGFbeta1 (10 ng/ml) treatment, a significantly increased number of DoHH2 cells was retained in G(0)/G(1) phase. We examined the level of cell cycle components, cyclins, cyclin-dependent kinases (cdk), and their inhibitors. We found that the expression of cyclin A and p21(WAF1) molecules was primarily modulated by TGFbeta1 treatment while the expression of other regulatory components, like cyclins D, cyclin E, cdk2, cdk4, and cdk6 or p15(INK4B), p16(INK4A), and p27(KIP1) was not significantly affected. We further examined expression and activity of CREB/ATF family members to examine their roles in cyclin A inhibition. The binding activity of CREB-1 and ATF-2 to the CRE region of the cyclin A promoter was almost completely abolished due to the treatment. The total level of CREB-1, ATF-2, and ATF-3 was notably reduced. Moreover, CREB-1 was dephosphorylated due to the treatment as revealed by immunoblotting. We assume that down-regulation of cyclin A was mediated by the absence of CREB/ATF activation dimers. The profound effect on the ATF family of transcription factors indicates the complexity of TGFbeta1 action on FL B malignant cells.

Topics: Cell Cycle; Cell Division; Cyclin A; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinases; Cyclins; DNA-Binding Proteins; G1 Phase; Gene Expression Regulation, Neoplastic; Humans; Kinetics; Lymphoma, B-Cell; Lymphoma, Follicular; Resting Phase, Cell Cycle; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Tumor Cells, Cultured

The pattern of immunoreactivity for amyloid precursor (APP) and B-cell lymphoma protooncogene (Bcl-2) proteins in the rat retina was studied after intravitreal injection of basic fibroblast growth factor (bFGF) or transforming growth factor-beta (TGFbeta). In normal control retinas, intense immunostaining of APP and Bcl-2 was observed primarily in the endfeet and proximal part of radial processes of Müller glial cells. A dose-dependent reduction in immunostaining of APP and Bcl-2 in Müller cells was observed after injection of bFGF and TGFbeta. These results provide the first evidence that APP and Bcl-2 can be down-regulated by cytokines in vivo.

Topics: Amyloid beta-Protein Precursor; Animals; Dose-Response Relationship, Drug; Down-Regulation; Fibroblast Growth Factor 2; Immunohistochemistry; Injections; Lymphoma, B-Cell; Neuroglia; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Retina; Transforming Growth Factor beta; Vitreous Body

Transcriptional regulation can occur at the level of initiation and RNA elongation. We report that the rearranged, nontranscribed Ig kappa gene in the pre-B cell line 70Z/3 harbors a paused RNA polymerase II (pol II) at a position between 45 and 89 bp downstream of the transcription initiation site. LPS, an inducer of NF-kappa B, activated Ig kappa gene transcription by increasing the processivity of pol II. TGF-beta inhibited the LPS-induced transcription of the Ig kappa gene, but not initiation and pausing of pol II. A rearranged copy of the Ig kappa gene was introduced into 70Z/3 cells using an episomal vector system. The episomal Ig kappa was regulated by LPS and TGF-beta like the endogenous gene and established a paused pol II, whereas a construct with a deletion of the intron enhancer and the C region did not establish a paused pol II. Two distinct functions can therefore be assigned to the deleted DNA elements: loading of pol II to its pause site and induction of processive transcription upon LPS stimulation. It had been proposed that somatic hypermutation of Ig genes is connected to transcription. The pause site of pol II described in this work resides upstream of the previously defined 5' boundary of mutator activity at Ig kappa genes. The possible role of pausing of pol II for somatic hypermutation is discussed.

Topics: Animals; B-Lymphocytes; Base Sequence; Gene Expression Regulation; Gene Rearrangement, B-Lymphocyte, Light Chain; Genes, Immunoglobulin; Genetic Vectors; Humans; Immunoglobulin kappa-Chains; Lymphoma, B-Cell; Mice; Molecular Sequence Data; NF-kappa B; Promoter Regions, Genetic; RNA Polymerase II; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

TGF-beta1 inhibits the cell cycle progression of many types of cells by arresting them in the G1 phase. This cell cycle arrest has been attributed to the regulatory effects of TGF-beta1 on both the levels and the activities of the G1 cyclins and their kinase partners. The activities of these kinases are negatively regulated by a number of proteins, such as p15INK4b, p21WAF1/Cip1, and p27Kip1, that physically associate with cyclins, cyclin-dependent kinases (Cdk), or cyclin-Cdk complexes. In epithelial cell lines, TGF-beta1 was previously shown to inhibit cell cycle progression through down-regulation of Cdk4 and/or up-regulation of p15INK4b and/or p21WAF1/Cip1. However, TGF-beta1 had little or no effect on the p27Kip1 mRNA and protein levels. In this report, we show that, in contrast to observations in epithelial cell lines, TGF-beta1 increased the p27Kip1 mRNA and protein levels in the murine B cell lines CH31 and WEHI231. This TGF-beta1-mediated induction of p27Kip1 also resulted in an increased association of p27Kip1 with Cdk2 and a decreased Cdk2 kinase activity. In contrast to epithelial cells, however, TGF-beta1 had little or no effect on the Cdk4 and p21WAF1/Cip1 protein levels in these B cells. Finally, although several studies suggested a direct role of p53 in TGF-beta1-mediated cell cycle arrest in epithelial cells, TGF-beta1 inhibited cell cycle progression in CH31 even in the absence of wild-type p53. Taken together, these results suggest that TGF-beta1 induces G1 arrest in B cells primarily through a p53-independent up-regulation of p27Kip1 protein.

Topics: Animals; B-Lymphocytes; Carrier Proteins; CDC2-CDC28 Kinases; Cell Cycle Proteins; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p15; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclin-Dependent Kinases; Cyclins; Enzyme Activation; Lymphoma, B-Cell; Mice; Microtubule-Associated Proteins; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; RNA, Messenger; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Up-Regulation

TGF-beta1 directs class switching to IgA by splenic B cells and by the surface IgM+ B cell line, I.29mu, by inducing germline (GL) Ig alpha transcripts. The promoter segment between -130 and +46, relative to the first initiation site for mouse GL alpha transcripts, is sufficient for expression and TGF-beta1 inducibility of a reporter gene in B cell lines. Within this segment resides a TGF-beta1-responsive element (TbetaRE) that is required for induction of the promoter by TGF-beta1 and, when multimerized, is sufficient to transfer TGF-beta1 inducibility to another promoter. In this report we show that a TGF-beta1-inducible complex binds the TbetaRE and contains the transcription factor core-binding factor (CBF; also known as acute myeloid leukemia, AML). Although all three CBF alpha family members activate the GL alpha promoter, only CBF alpha3 (AML-2) is induced by TGF-beta1 in splenic B and I.29mu cells. The TbetaRE contains two CBF binding sites. Mutation of both sites reduces but does not eliminate induction of the GL alpha promoter by TGF-beta1 or by overexpression of CBF, possibly due to the presence of an additional CBF site in the promoter. In addition, the TbetaRE contains two copies of another sequence motif. Mutation of these motifs eliminates TGF-beta1 induction of the GL alpha promoter. Together the data indicate that TGF-beta1 induction of the alpha promoter involves induction of CBF alpha3, which binds to the TbetaRE of the promoter along with one or more proteins.

Topics: Animals; B-Lymphocytes; Core Binding Factors; DNA-Binding Proteins; Gene Expression Regulation; Genes, Immunoglobulin; Genes, Reporter; Immunoglobulin A; Immunoglobulin Class Switching; Immunoglobulin M; Lymphoma, B-Cell; Macromolecular Substances; Mice; Neoplasm Proteins; Promoter Regions, Genetic; Receptors, Antigen, B-Cell; Spleen; Transcription Factors; Transforming Growth Factor beta; Tumor Cells, Cultured

Malignant lymphoma frequently develops in the pleural cavity of the patients with long-standing pyothorax. Thus, the term pyothorax-associated lymphoma (PAL) has been proposed for this type of tumor. Most of PALs are diffuse lymphoma of B cell type and contain Epstein-Barr virus (EBV) DNA. We have established two lymphoma cell lines from the biopsy specimens of PAL cases, OPL-1 and OPL-2. Both cell lines contain EBV DNA, but only OPL-1 expresses Epstein-Barr virus nuclear antigen 2 (EBNA2) that works as a target molecule for cell-mediated immune response. In this study, we examined the expression of immunosuppressive factors in OPLs. Only OPL-1, not OPL-2, expressed interleukin-10 (IL-10) mRNA and secreted IL-10 into culture supernatant. Both OPL-1 and OPL-2 expressed transforming growth factor (TGF) beta 1 mRNA, however, neither expressed latent TGF beta binding protein (LTBP) mRNA at detectable level by Northern blot analysis. Because TGF beta expresses its functions in cooperation with LTBP, the biological functions of TGF beta 1 could be negligible. Neither cell lines expressed EBV BCRF1 mRNA at detectable level, a viral gene product which is partly homologous to human IL-10 and shares biological activities of IL-10. Since OPL-1 shows weaker proliferative activity than OPL-2 and expresses viral antigens, the production of an immunosuppressive cytokine, IL-10, might contribute to the development of overt lymphoma. The present study suggested that immunosuppressive cytokine plays a role in lymphomagenesis of immunocompetent patients.

Topics: DNA, Viral; Empyema, Pleural; Epstein-Barr Virus Nuclear Antigens; Herpesvirus 4, Human; Humans; Immune Tolerance; Interleukin-10; Lymphoma, B-Cell; Pleural Neoplasms; Proliferating Cell Nuclear Antigen; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

We have developed an efficient in vitro class switching system using a subclone (CH12F3) of the IgM+ CH12.LX lymphoma cell line. CH12F3 cells switched from surface IgM+ cells to surface IgA+ cells at a high frequency (50%) after 72 h stimulation with IL-4, transforming growth factor (TGF)-beta and CD40L. No other class isotype-producing cells were detected, indicating that the CH12F3 clone is exclusively committed to IgA isotype switching. To understand the molecular basis of the isotype commitment, we studied the methylation profiles of I region promoters and I region transcription of CH12F3 cells. No germline transcripts other than those from the I alpha region were detected and only the I alpha promoter was demethylated in uninduced CH12F3 cells. TGF-beta, CD40L and IL-4 synergistically induced efficient switch recombination in CH12F3 cells, suggesting that the three stimulations up-regulate different steps of switch recombination in isotype-committed B cells such as CH12F3 cells. Stimulation of CH12F3 cells by IL-4 or TGF-beta, but not by CD40L, induced transient but complete methylation of the I alpha region. TGF-beta and CD40L, but not IL-4, increased the amounts of germline alpha transcripts. We found that the extents of methylation and the amounts of germline transcripts do not necessarily correlate with the efficiency of recombination in induced CH12F3 cells. These results led to the proposal that switch recombination can be separated into at least two phases, i.e. commitment and recombination. The roles of IL-4, TGF-beta and CD40L in the two phases are discussed.

Topics: Animals; B-Lymphocytes; CD40 Antigens; CD40 Ligand; Cell Separation; Clone Cells; DNA Methylation; Female; Immunoglobulin A; Immunoglobulin Class Switching; Immunoglobulin M; Interleukin-4; Ligands; Lymphocyte Activation; Lymphoma, B-Cell; Membrane Glycoproteins; Mice; Transcription, Genetic; Transforming Growth Factor beta; Tumor Cells, Cultured

Vitamin E succinate (VES) and all-trans-retinoic acid (RA) were determined to be growth inhibitory for B lymphoma cells in vitro. RL, an Epstein-Barr virus-negative human cell line, was growth suppressed 87% with VES (5 micrograms/ml) and 58% with RA (10(-6) M); both agents blocked the cells in G1 of the cell cycle. The antiproliferative effect of VES seems to be independent of its potential antioxidant property because both fat- and water-soluble antioxidants were found to have no effect on RL cell proliferation. VES and RA increased IgM antibody concentrations in cell supernatants 5.8- and 9.9-fold, respectively. DNA fragmentation and flow cytometry studies showed VES- and RA-induced apoptosis in RL cells. VES- and RA-treated RL cells gradually underwent apoptosis over time with maximal induction occurring at days 6 and 5 of culture, respectively. A role for transforming growth factor beta in VES- and RA-mediated RL growth suppression is indicated by increased ligand and type II receptor protein expression. Furthermore, neutralizing antibodies to transforming growth factor beta 1 partially blocked the growth suppressive action of both VES and RA, thus suggesting that a TGF-beta autocrine negative loop was involved in VES and RA suppression of RL cell growth.

Topics: Antibodies, Neoplasm; Antioxidants; Apoptosis; Cell Division; G1 Phase; Humans; Immunoglobulin M; Lymphoma, B-Cell; Receptors, Antigen, B-Cell; Tocopherols; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured; Vitamin E

We examined the effects of inflammatory cytokines, such as interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF alpha), transforming growth factor-beta (TGF beta) and lipopolysaccharide (LPS), on the urokinase-type plasminogen activator (uPA) gene expression in RC-K8 human pre-B lymphoma cells. Recombinant IL-1 alpha, recombinant IL-1 beta and LPS but not recombinant IL-6, recombinant TNF alpha and TGF beta dose-dependently increased uPA accumulation in the conditioned medium. Northern blot analysis revealed that uPA mRNA levels rapidly increased with a peak induction at 2 h after stimulation with IL-1 alpha and IL- 1 beta, but uPA mRNA increase by LPS began at 9 h after stimulation and the increase was maintained until the experiment ended at 24 h. These responses were independent of de novo synthesis, rather amplified in the presence of a protein synthesis inhibitor. The effects by IL-1 alpha and Il-1 beta were prevented by addition of anti-IL-1 alpha and anti-IL-1 beta neutralizing antibodies, respectively. In contrast, both antibodies did not prevent LPS-induced uPA gene expression. Therefore, it is unlikely that the effect by LPS is through induction of IL-1. Both IL-1 alpha and IL- 1 beta rapidly activated uPA gene transcription, but not increased stability of uPA mRNA. These results suggest that both IL-1 alpha and IL-1 beta cause a rapid activation of uPA gene transcription in which de novo protein synthesis is not required and that LPS induces uPA gene expression independently of the IL-1 pathway. These modulations of uPA production by inflammatory mediators may be implicated in tumor growth and metastasis.

Topics: Antigen-Antibody Reactions; Cells, Cultured; Cytokines; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Interleukin-1; Interleukin-6; Lipopolysaccharides; Lymphoma, B-Cell; Precancerous Conditions; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Urokinase-Type Plasminogen Activator

A patient with chronic lymphocytic leukemia developed a large cell lymphoma apparently derived from the same neoplastic B-cell clone (Richter's syndrome). At the same time, mitogen-stimulated proliferation of the patient's circulating leukemic B-cells was no longer inhibited by the regulatory cytokine transforming growth factor-beta (TGF-beta), suggesting that such loss of inhibition might be contributing to the clinical and biological progression of the disease.

Topics: Aged; B-Lymphocytes; Cell Division; Depression, Chemical; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Lymphoma, Large B-Cell, Diffuse; Male; Syndrome; Transforming Growth Factor beta; Tumor Cells, Cultured

Treatment of the WEHI-2131 or CH31 B cell lymphomas with anti-mu or transforming growth factor (TGF)-beta leads to growth inhibition and subsequent cell death via apoptosis. Since anti-mu stimulates a transient increase in c-myc and c-fos transcription in these lymphomas, we examined the role of these proteins in growth regulation using antisense oligonucleotides. Herein, we demonstrate that antisense oligonucleotides for c-myc prevent both anti-mu- and TGF-beta-mediated growth inhibition in the CH31 and WEHI-231 B cell lymphomas, whereas antisense c-fos has no effect. Furthermore, antisense c-myc promotes the appearance of phosphorylated retinoblastoma protein in the presence of anti-mu and prevents the progression to apoptosis as measured by propidium iodide staining. Northern and Western analyses show that c-myc message and the levels of multiple myc proteins were maintained in the presence of antisense c-myc, results indicating that myc species are critical for the continuation of proliferation and the prevention of apoptosis. These data implicate c-myc in the negative signaling pathway of both TGF-beta and anti-mu.

Topics: Animals; Apoptosis; B-Lymphocytes; Base Sequence; Cell Cycle; Cell Division; Immune Tolerance; Immunoglobulin mu-Chains; Lymphocyte Activation; Lymphoma, B-Cell; Mice; Molecular Sequence Data; Oligonucleotides, Antisense; Phosphorylation; Proto-Oncogene Proteins c-myc; Retinoblastoma Protein; Transforming Growth Factor beta; Tumor Cells, Cultured

Topics: Animals; Antibodies; Antigens, CD; CD5 Antigens; Cell Division; Immunoglobulin M; Kinetics; Lymphoma, B-Cell; Mice; Transforming Growth Factor beta; Tumor Cells, Cultured

Cross-linking of B-cell membrane immunoglobulin (Ig) receptors induces growth arrest at G1-S, leading to apoptosis and cell death in the immature lymphomas WEHI-231 and CH31, but not in the CH12 B-cell line. In this system, which has been used as a model for B-cell tolerance, we have established that these lymphomas produce active transforming growth factor beta (TGF-beta) when treated with anti-Ig and that their hierarchy of sensitivity to TGF-beta generally correlates with their growth inhibition by anti-Ig. TGF-beta, in turn, has been shown to interfere with the phosphorylation of the retinoblastoma gene product, pRB. Herein, we also demonstrate that in WEHI-231 B-lymphoma cells treated with anti-Ig for 24 h, the pRB protein is found to be predominantly in the underphosphorylated form, as previously reported for cells arrested by the exogenous addition of TGF-beta. However, neutralizing antibodies to TGF-beta failed to prevent growth inhibition by anti-Ig in WEHI-231 and CH31. When WEHI-231 lymphoma cells were selected for growth in TGF-beta, the majority of the TGF-beta-resistant clones remained sensitive to anti-Ig-mediated growth inhibition. In these clones, the retinoblastoma gene product was found to be in the underphosphorylated form after 24-h treatment with anti-Ig, but not with TGF-beta. These data show that anti-Ig treatment of murine B-cell lymphomas stimulates the production of active TGF-beta but that a TGF-beta-independent pathway may be responsible for the pRB underphosphorylation and cell cycle blockade.

Topics: Animals; Antibodies, Anti-Idiotypic; Cell Death; Cell Division; Lymphoma, B-Cell; Mice; Phosphorylation; Retinoblastoma Protein; Transforming Growth Factor beta; Tumor Cells, Cultured

Cytokines such as interleukin-5 (IL-5) and transforming growth factor beta 1 (TGF beta 1) increase IgA production by heterogeneous populations of lipopolysaccharide (LPS)-activated murine B cells. We have used IgA expressing murine B-lymphoma cells CH12.LX.C4.4F10 (4F10) to define the activity of these and other cytokines on IgA secretion at the single-cell level, membrane IgA expression, IgA polymerization and cell growth. IL-5 as well as LPS significantly increases IgA secretion of 4F10 cells, whereas TGF beta 1, a cytokine known to stimulate isotype switching to IgA among surface IgM-bearing B cells, inhibits IgA secretion. When tested alone, IL-1 beta, IL-2, IL-4, IL-6 and interferon-gamma (IFN-gamma) do not significantly alter IgA secretion. However, there is a synergistic increase in IgA secretion when 4F10 cells are co-stimulated with IL-5 and IL-4, while IFN-gamma inhibits IL-5-stimulated up-regulation of IgA secretion. In parallel with increased IgA secretion after cytokine stimulation, 4F10 cells display less membrane IgA. Increased J-chain steady-state mRNA levels after IL-5 or LPS stimulation are paralleled by increased mRNA levels for secreted IgA, but are not accompanied by alterations in the ratio of monomeric to polymeric IgA. IL-5 and LPS initially stimulated but later inhibited 4F10 cell proliferation suggesting an inverse relationship between proliferation and differentiation in this cell line. 4F10 cells are a useful model for the characterization of discrete aspects of IgA B-cell differentiation, since the secretory and membrane Ig and proliferative responses of this IgA B-cell line to cytokines and LPS appear to parallel those of freshly isolated murine B cells.

Topics: Animals; Antibodies, Neoplasm; B-Lymphocytes; Blotting, Northern; Cell Differentiation; Cell Line; Cytokines; Immunoglobulin A; Interleukin-5; Lipopolysaccharides; Lymphoma, B-Cell; Mice; Transforming Growth Factor beta; Tumor Cells, Cultured

H chain isotype switch recombination is preceded by the appearance of RNA initiating 5' of the specific switch region that will undergo recombination. In an effort to understand the potential function of germline transcripts in switch recombination and whether the regulation of germline transcripts correlates with the regulation of switching, we are studying this process in the murine B lymphoma cell line I.29 mu, which switches, after treatment with bacterial LPS, primarily to IgA and less frequently to IgE. Levels of alpha germline transcripts initiating upstream of alpha switch (S alpha) sequences are elevated in clones of this line that switch well, compared with clones that switch less frequently. Transforming growth factor-beta (TGF-beta) has been shown to increase alpha germline transcripts and switching to IgA expression in LPS-stimulated murine splenic B cells. We now demonstrate that TGF-beta increases LPS-induced switching to IgA by 10-fold at optimal doses and increases the level of alpha germline transcripts 5- to 9-fold in I.29 mu cells. Nuclear run-on analysis shows that this increase is at the level of transcription. Thus, TGF-beta appears to direct switching to IgA by inducing transcription from the unrearranged S alpha-C alpha DNA segment. Germline alpha RNA is quite stable in I.29 mu cells, having a half-life of about 5 h, and we find no evidence for further stabilization in the presence of TGF-beta. Levels of epsilon germline transcripts are decreased by TGF-beta treatment. IL-4, which modestly increases switching in I.29 mu cells, slightly increases transcription of alpha germline RNA. IFN-gamma, which reduces switching to IgA in these cells, also reduces the level of alpha germline transcripts. IFN-gamma also reduces the level of epsilon germline transcripts induced by IL-4. Our results support the hypothesis that the regulation of transcription of particular switch sequences by cytokines regulates the specificity of recombination. We also present evidence that IL-4 may provide other signals, distinct from transcriptional targeting, that increase LPS-induced switching to IgA.

Topics: Animals; Cells, Cultured; Genes, Immunoglobulin; Immunoglobulin A; Immunoglobulin alpha-Chains; Immunoglobulin Constant Regions; Immunoglobulin Switch Region; Interferon-gamma; Interleukin-4; Lipopolysaccharides; Lymphoma, B-Cell; Mice; Transcription, Genetic; Transforming Growth Factor beta

Exposure of the murine B lymphoma cell line WEHI-231 to anti-immunoglobulin M (anti-IgM) antibodies results in growth arrest in the G1 phase of the cell cycle followed by programmed cell death. This response may be analogous to the clonal deletion of immature B cells that occurs when the membrane IgM on these cells is engaged by self-antigens or by anti-IgM antibodies. Thus the WEHI-231 cell line has been a useful in vitro system for identifying factors that modulate anti-Ig-induced growth inhibition and/or clonal deletion. For example, both antigen-induced tolerance induction in immature B cells and anti-Ig-induced growth arrest of WEHI-231 cells are prevented by bacterial lipopolysaccharide or by the products of activated T helper cells. Since negative signaling by membrane Ig may also be regulated by additional factors, we asked whether other cytokines or hormones would regulate the growth of WEHI-231 cells or its response to anti-IgM antibodies. We show here that two compounds that are generally immunosuppressive, transforming growth factor beta 1 (TGF-beta 1) and the synthetic corticosteroid, dexamethasone, blocked the ability of lipopolysaccharide and T cell-derived lymphokines to protect WEHI-231 cells from anti-IgM-induced growth arrest. In addition, TGF-beta 1 and dexamethasone slightly inhibited the growth of WEHI-231 cells by themselves and also potentiated the growth inhibitory effects of anti-IgM antibodies. Thus for WEHI-231 cells, TGF-beta 1 and dexamethasone are inhibitory factors which favor growth arrest.

Topics: Animals; Antibodies, Anti-Idiotypic; B-Lymphocytes; Cell Differentiation; Cell Division; Dexamethasone; Lipopolysaccharides; Lymphokines; Lymphoma, B-Cell; Transforming Growth Factor beta; Tumor Cells, Cultured