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1.
Mol Cell Biol ; 21(14): 4515-27, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11416131

ABSTRACT

The interaction of interleukin-2 (IL-2) with its receptor (IL-2R) critically regulates the T-cell immune response, and the alpha chain CD25/IL-2Ralpha is required for the formation of the high-affinity receptor. Tissue-specific, inducible expression of the IL-2Ralpha gene is regulated by at least three positive regulatory regions (PRRI, PRRII, and PRRIII), but none responded to CD28 engagement in gene reporter assays although CD28 costimulation strongly amplifies IL-2Ralpha gene transcription. By DNase I hypersensitivity analysis, we have identified a novel TCR-CD3- and CD28-responsive enhancer (CD28rE) located 8.5 kb 5' of the IL-2Ralpha gene. PRRIV/CD28rE contains a functional CRE/TRE element required for CD28 signaling. The T-cell-specific, CD28-responsive expression of the IL-2Ralpha gene appears controlled through PRRIV/CD28rE by cooperation of CREB/ATF and AP-1 family transcription factors.


Subject(s)
Blood Proteins/metabolism , CD28 Antigens/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Enhancer Elements, Genetic , Receptors, Interleukin-2/genetics , Transcription Factor AP-1/metabolism , Transcription Factors/metabolism , Transcriptional Activation , Activating Transcription Factors , Animals , Base Sequence , COS Cells , Chlorocebus aethiops , DNA, Complementary , Humans , Jurkat Cells , Molecular Sequence Data , Receptor-CD3 Complex, Antigen, T-Cell/metabolism , Regulatory Sequences, Nucleic Acid
2.
Curr Biol ; 11(8): 579-86, 2001 Apr 17.
Article in English | MEDLINE | ID: mdl-11369202

ABSTRACT

BACKGROUND: Mammalian megakaryocytes release blood platelets through a remarkable process of cytoplasmic fragmentation and de novo assembly of a marginal microtubule band. Cell-specific components of this process include the divergent beta-tubulin isoform beta1 that is expressed exclusively, and is the predominant isoform, in platelets and megakaryocytes. The functional significance of this restricted expression, and indeed of the surprisingly large repertoire of metazoan tubulin genes, is unclear. Fungal tubulin isoforms appear to be functionally redundant, and all mammalian beta-tubulins can assemble in a variety of microtubules, whereas selected fly and worm beta-tubulins are essential in spermatogenesis and neurogenesis. To address the essential role of beta1-tubulin in its natural context, we generated mice with targeted gene disruption. RESULTS: beta1-tubulin(-/-) mice have thrombocytopenia resulting from a defect in generating proplatelets, the immediate precursors of blood platelets. Circulating platelets lack the characteristic discoid shape and have defective marginal bands with reduced microtubule coilings. beta1-tubulin(-/-) mice also have a prolonged bleeding time, and their platelets show an attenuated response to thrombin. Two alternative tubulin isoforms, beta2 and beta5, are overexpressed, and the total beta-tubulin content of beta1-tubulin(-/-) megakaryocytes is normal. However, these isoforms assemble much less efficiently into platelet microtubules and are thus unable to compensate completely for the absence of beta1-tubulin. CONCLUSIONS: This is the first genetic study to address the essential functions of a mammalian tubulin isoform in vivo. The results establish a specialized role for beta1-tubulin in platelet synthesis, structure, and function.


Subject(s)
Blood Platelets/physiology , Tubulin/physiology , Animals , Blood Platelets/cytology , Cell Lineage , Mice , Mice, Knockout , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Isoforms/physiology , Thrombocytopenia/etiology , Tubulin/genetics , Tubulin/metabolism
3.
J Immunol ; 165(10): 5874-83, 2000 Nov 15.
Article in English | MEDLINE | ID: mdl-11067948

ABSTRACT

Peripheral T lymphocyte activation in response to TCR/CD3 stimulation is reduced in type 1 diabetic patients. To explore the basis of this deficiency, a comprehensive analysis of the signal transduction pathway downstream of the TCR/CD3 complex was performed for a cohort of patients (n = 38). The main result of the study shows that T cell hyporesponsiveness is positively correlated with a reduced amount of p56(lck) in resting T lymphocytes. Upon CD3-mediated activation, this defect leads to a hypophosphorylation of the CD3zeta-chain and few other polypeptides without affecting the recruitment of ZAP70. Other downstream effectors of the TCR/CD3 transduction machinery, such as phosphatidylinositol 3-kinase p85alpha, p59(fyn), linker for activation of T cells (LAT), and phospholipase C-gamma1, are not affected. In some patients, the severity of this phenotypic deficit could be linked to low levels of p56(lck) mRNA and resulted in the failure to efficiently induce the expression of the CD69 early activation marker. We propose that a primary deficiency in human type 1 diabetes is a defect in TCR/CD3-mediated T cell activation due to the abnormal expression of the p56(lck) tyrosine kinase.


Subject(s)
Diabetes Mellitus, Type 1/enzymology , Diabetes Mellitus, Type 1/genetics , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/biosynthesis , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/deficiency , T-Lymphocyte Subsets/enzymology , Adolescent , Adult , Cohort Studies , Female , Flow Cytometry , Humans , Immune Tolerance/genetics , Interphase/genetics , Interphase/immunology , Leukocytes, Mononuclear/enzymology , Leukocytes, Mononuclear/immunology , Lymphocyte Activation/genetics , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Male , Middle Aged , Phosphorylation , Phosphotyrosine/metabolism , RNA, Messenger/biosynthesis , Receptor-CD3 Complex, Antigen, T-Cell/physiology , T-Lymphocyte Subsets/metabolism
4.
Blood ; 96(4): 1366-73, 2000 Aug 15.
Article in English | MEDLINE | ID: mdl-10942379

ABSTRACT

The cellular and molecular bases of platelet release by terminally differentiated megakaryocytes represent important questions in cell biology and hematopoiesis. Mice lacking the transcription factor NF-E2 show profound thrombocytopenia, and their megakaryocytes fail to produce proplatelets, the microtubule-based precursors of blood platelets. Using mRNA subtraction between normal and NF-E2-deficient megakaryocytes, cDNA was isolated encoding beta1 tubulin, the most divergent beta tubulin isoform. In NF-E2-deficient megakaryocytes, beta1 tubulin mRNA and protein are virtually absent. The expression of beta1 tubulin is exquisitely restricted to platelets and megakaryocytes, where it appears late in differentiation and localizes to microtubule shafts and coils within proplatelets. Restoring NF-E2 activity in a megakaryoblastic cell line or in NF-E2-deficient primary megakaryocytes rescues the expression of beta1 tubulin. Re-expressing beta1 tubulin in isolation does not, however, restore proplatelet formation in the defective megakaryocytes, indicating that other critical factors are required; indeed, other genes identified by mRNA subtraction also encode structural and regulatory components of the cytoskeleton. These findings provide critical mechanistic links between NF-E2, platelet formation, and selected microtubule proteins, and they also provide novel molecular insights into thrombopoiesis. (Blood. 2000;96:1366-1373)


Subject(s)
Blood Platelets/cytology , Blood Platelets/physiology , Cell Lineage/physiology , DNA-Binding Proteins/physiology , Transcription Factors/physiology , Tubulin/physiology , Animals , Cell Differentiation/physiology , Erythroid-Specific DNA-Binding Factors , Hematopoiesis/physiology , Mice , NF-E2 Transcription Factor , NF-E2 Transcription Factor, p45 Subunit , Nuclear Proteins/physiology , RNA, Messenger/biosynthesis , Repressor Proteins/physiology
5.
Oncogene ; 19(17): 2086-97, 2000 Apr 20.
Article in English | MEDLINE | ID: mdl-10815800

ABSTRACT

Activation of Stat5 by many cytokines implies that it cannot alone insure the specificity of the regulation of its target genes. We have evidenced a physical and functional interaction between members of two unrelated transcription factor families, Ets-1, Ets-2 and Stat5, which could contribute to the proliferative response to interleukin 2. Competition with GAS- and EBS-specific oligonucleotides and immunoassays with a set of anti-Stat and anti-Ets families revealed that the IL-2-induced Stat5-Ets complex recognizes several GAS motifs identified as target sites for activated Stat5 dimers. Coimmunoprecipitation experiments evidenced that a Stat5/Ets-1/2 complex is formed in vivo in absence of DNA. GST-pull down experiments demonstrated that the C-terminal domain of Ets-1 is sufficient for this interaction in vitro. Cotransfection experiments in Kit225 T cells resulted in cooperative transcriptional activity between both transcription factors in response to a combination of IL-2, PMA and ionomycin. A Stat5-Ets protein complex was the major inducible DNA-binding complex bound to the human IL-2rE GASd/EBSd motif in long-term proliferating normal human T cells activated by CD2 and CD28. These results suggest that the inducible Stat5-Ets protein interaction plays a role in the regulation of gene expression in response to IL-2 in human T lymphocytes.


Subject(s)
DNA-Binding Proteins/metabolism , Interleukin-2/metabolism , Milk Proteins , Proto-Oncogene Proteins/metabolism , Repressor Proteins , T-Lymphocytes/physiology , Trans-Activators/metabolism , Transcription Factors/metabolism , DNA-Binding Proteins/genetics , Humans , Immune Sera , Interleukin-2/pharmacology , Lymphocyte Activation , Mitogens/pharmacology , Proto-Oncogene Protein c-ets-1 , Proto-Oncogene Protein c-ets-2 , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/immunology , Proto-Oncogene Proteins c-ets , Regulatory Sequences, Nucleic Acid , STAT5 Transcription Factor , T-Lymphocytes/drug effects , Trans-Activators/genetics , Trans-Activators/immunology , Transcription Factors/genetics , Transcription Factors/immunology , Transcriptional Activation , Transfection
6.
J Cell Biol ; 147(6): 1299-312, 1999 Dec 13.
Article in English | MEDLINE | ID: mdl-10601342

ABSTRACT

Megakaryocytes release mature platelets in a complex process. Platelets are known to be released from intermediate structures, designated proplatelets, which are long, tubelike extensions of the megakaryocyte cytoplasm. We have resolved the ultrastructure of the megakaryocyte cytoskeleton at specific stages of proplatelet morphogenesis and correlated these structures with cytoplasmic remodeling events defined by video microscopy. Platelet production begins with the extension of large pseudopodia that use unique cortical bundles of microtubules to elongate and form thin proplatelet processes with bulbous ends; these contain a peripheral bundle of microtubules that loops upon itself and forms a teardrop-shaped structure. Contrary to prior observations and assumptions, time-lapse microscopy reveals proplatelet processes to be extremely dynamic structures that interconvert reversibly between spread and tubular forms. Microtubule coils similar to those observed in blood platelets are detected only at the ends of proplatelets and not within the platelet-sized beads found along the length of proplatelet extensions. Growth and extension of proplatelet processes is associated with repeated bending and bifurcation, which results in considerable amplification of free ends. These aspects are inhibited by cytochalasin B and, therefore, are dependent on actin. We propose that mature platelets are assembled de novo and released only at the ends of proplatelets, and that the complex bending and branching observed during proplatelet morphogenesis represents an elegant mechanism to increase the numbers of proplatelet ends.


Subject(s)
Blood Platelets/cytology , Blood Platelets/metabolism , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Megakaryocytes/cytology , Megakaryocytes/metabolism , Actins/metabolism , Animals , Blood Platelets/drug effects , Blood Platelets/ultrastructure , Cell Differentiation/drug effects , Cell Size/drug effects , Cells, Cultured , Cytochalasin B/pharmacology , Cytoplasm/drug effects , Cytoplasm/metabolism , Cytoplasm/ultrastructure , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/ultrastructure , Megakaryocytes/drug effects , Megakaryocytes/ultrastructure , Mice , Microscopy, Electron , Microscopy, Video , Microtubules/drug effects , Microtubules/metabolism , Microtubules/ultrastructure , Models, Biological , Nocodazole/pharmacology , Pseudopodia/drug effects , Pseudopodia/metabolism , Pseudopodia/ultrastructure , Time Factors
7.
Blood ; 94(9): 3037-47, 1999 Nov 01.
Article in English | MEDLINE | ID: mdl-10556187

ABSTRACT

Expression of the p45 subunit of transcription factor NF-E2 is restricted to selected blood cell lineages, including megakaryocytes and developing erythrocytes. Mice lacking p45 NF-E2 show profound thrombocytopenia, resulting from a late arrest in megakaryocyte differentiation, and a number of red blood cell defects, including anisocytosis and hypochromia. Here we report results of studies aimed to explore the pathophysiology of these abnormalities. Mice lacking NF-E2 produce very few platelet-like particles that display highly disorganized ultrastructure and respond poorly to platelet agonists, features consistent with the usually lethal hemorrhage in these animals. Thrombocytopenia was evident during fetal life and was not corrected by splenectomy in adults. Surprisingly, fetal NF-E2-deficient megakaryocyte progenitors showed reduced proliferation potential in vitro. Thus, NF-E2 is required for regulated megakaryocyte growth as well as for differentiation into platelets. All the erythroid abnormalities were reproduced in lethally irradiated wild-type recipients of hematopoietic cells derived from NF-E2-null fetuses. Whole blood from mice lacking p45 NF-E2 showed numerous small red blood cell fragments; however, survival of intact erythrocytes in vivo was indistinguishable from control mice. Considered together, these observations indicate a requirement for NF-E2 in generating normal erythrocytes. Despite impressive splenomegaly at baseline, mice lacking p45 NF-E2 survived splenectomy, which resulted in increased reticulocyte numbers. This reveals considerable erythroid reserve within extra-splenic sites of hematopoiesis and suggests a role for the spleen in clearing abnormal erythrocytes. Our findings address distinct aspects of the requirements for NF-E2 in blood cell homeostasis and establish its roles in proper differentiation of megakaryocytes and erythrocytes.


Subject(s)
Anemia/genetics , DNA-Binding Proteins/genetics , Thrombocytopenia/genetics , Transcription Factors/genetics , Anemia/physiopathology , Animals , Cell Lineage/genetics , Disease Models, Animal , Erythroid-Specific DNA-Binding Factors , Erythropoiesis/genetics , Gene Expression Regulation , Mice , Mice, Knockout , NF-E2 Transcription Factor , NF-E2 Transcription Factor, p45 Subunit , Thrombocytopenia/physiopathology
8.
Blood ; 92(5): 1608-16, 1998 Sep 01.
Article in English | MEDLINE | ID: mdl-9716588

ABSTRACT

Mechanisms of platelet production and release by mammalian megakaryocytes are poorly understood. We used thrombocytopenic knockout mice to better understand these processes. Proplatelets are filamentous extensions of terminally differentiated megakaryocytes that are thought to represent one mechanism of platelet release; however, these structures have largely been recognized in cultured cells and there has been no correlation between thrombocytopoiesis in vivo and proplatelet formation. Mice lacking transcription factor NF-E2 have a late arrest in megakaryocyte maturation, resulting in profound thrombocytopenia. In contrast to normal megakaryocytes, which generate abundant proplatelets, cells from these mice never produce proplatelets, even after prolonged stimulation with c-Mpl ligand. Similarly, megakaryocytes from thrombocytopenic mice with lineage-selective loss of transcription factor GATA-1 produce proplatelets very rarely. These findings establish a significant correlation between thrombocytopoiesis and proplatelet formation and suggest that the latter represents a physiologic mechanism of platelet release. We further show that proplatelet formation by normal megakaryocytes and its absence in cells lacking NF-E2 are independent of interactions with adherent (stromal) cells. Similarly, thrombocytopenia in NF-E2(-/-) mice reflects intrinsic defects in the megakaryocyte lineage. These observations improve our understanding of platelet production and validate the study of proplatelets in probing the underlying mechanisms.


Subject(s)
Blood Platelets/cytology , DNA-Binding Proteins/physiology , Hematopoiesis , Megakaryocytes/cytology , Transcription Factors/physiology , Animals , DNA-Binding Proteins/analysis , DNA-Binding Proteins/genetics , Erythroid-Specific DNA-Binding Factors , Fetal Tissue Transplantation , GATA1 Transcription Factor , Hematopoietic Stem Cells/chemistry , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/ultrastructure , Liver/embryology , Liver Transplantation , Mice , Mice, Inbred Strains , Mice, Knockout , Microscopy, Electron, Scanning , NF-E2 Transcription Factor , NF-E2 Transcription Factor, p45 Subunit , Thrombocytopenia/genetics , Transcription Factors/analysis , Transcription Factors/genetics
9.
J Biol Chem ; 273(13): 7572-8, 1998 Mar 27.
Article in English | MEDLINE | ID: mdl-9516460

ABSTRACT

Biochemical analysis of megakaryocytes, the precursors of blood platelets, is limited by their rarity in vivo, and studies on lineage-specific gene expression have been conducted exclusively in cell lines with limited megakaryocytic potential. Mice lacking the transcription factor NF-E2 display arrested megakaryocyte differentiation and profound thrombocytopenia. To study the heterodimeric NF-E2 protein in primary cells, we cultured mouse fetal livers with the c-Mpl ligand, obtained highly enriched megakaryocyte populations, and readily detected NF-E2 activity in nuclear extracts. As in erythroid cells, p45 NF-E2 is the only large subunit in primary megakaryocytes that dimerizes with distinct small Maf proteins to constitute a heterogeneous NF-E2 complex. Whereas p18/MafK is the predominant small Maf protein in erythroid cells, the related polypeptides MafG and/or MafF predominate in megakaryocytes. Although this represents the first example of differential small Maf protein expression among closely related blood lineages, the DNA-binding specificity of NF-E2 is similar in both cell types. Although the megakaryocyte protein preferentially binds an asymmetric AP-1-related motif, it also recognizes cAMP-responsive element-related sequences, albeit with lower affinity, and nucleotides outside the core sequence influence the DNA-protein interaction. These results demonstrate the feasibility of biochemical studies on primary murine megakaryocytes and provide a basis to dissect the critical functions of NF-E2 in megakaryocyte differentiation.


Subject(s)
DNA-Binding Proteins/metabolism , Megakaryocytes/metabolism , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Zinc Fingers , Animals , Base Sequence , Binding Sites , Cell Differentiation , Culture Techniques , DNA/chemistry , DNA/metabolism , Erythroid-Specific DNA-Binding Factors , Liver/embryology , MafK Transcription Factor , Megakaryocytes/cytology , Mice , NF-E2 Transcription Factor , NF-E2 Transcription Factor, p45 Subunit , Sequence Analysis, DNA , Thrombopoietin/metabolism , Tumor Cells, Cultured
10.
AIDS Res Hum Retroviruses ; 14(4): 353-65, 1998 Mar 01.
Article in English | MEDLINE | ID: mdl-9519897

ABSTRACT

X-irradiation has been used in the treatment of several human diseases, including AIDS-related-malignancies. X-irradiation might induce the transcription and the replication of human immunodeficiency virus type 1 (HIV-1) and enhance nuclear factor kappa B (NF-kappaB). In the present article we show that the activation of the HIV-1 long terminal repeat (LTR) by direct X-irradiation can be mimicked by coculture of transfected cells with X-irradiated nontransfected (HIV-1-negative) cells. In the human colonic carcinoma cell line HT29, the activation seems to depend on an extracellular factor(s) released by a cell line treated with X-rays. The HIV-1 LTR cis-acting element conferring X-indirect responsiveness was identified as the kappaB tandem motif. The two main nuclear HIV-1 kappaB-binding complexes activated by X-direct and -indirect irradiation were the NF-kappaB p50/p65 and c-Rel/p65 heterodimers. Nuclear NF-kappaB activation was dependent on protein neosynthesis. It was partially inhibited by 100 microM pyrrolidine dithiocarbamate, a potent antioxidant drug, but was not correlated with a significant decrease in cellular IkappaBalpha. Furthermore, X-irradiation induces the expression of several cytokine genes generally associated with stress response and antibodies against interleukin 6 and TNF-alpha partially inhibited the X-indirect activation of the HIV-1 LTR. The use of protein kinase C (PKC)-specific inhibitor and of forskolin, an adenylate cyclase activator, suggests that a PKC-dependent pathway and the cAMP intracellular concentration could play a role in the X-indirect enhancement of HIV-1 LTR transcription in the HT29 cell line. In addition, supernatants of an X-irradiated HT29 cell culture activated the HIV-1 stimulation in infected peripheral blood monocytes.


Subject(s)
HIV Long Terminal Repeat/radiation effects , HIV-1/genetics , HIV-1/radiation effects , NF-kappa B/metabolism , NF-kappa B/radiation effects , Antioxidants/pharmacology , Base Sequence , Binding Sites/genetics , Cell Line , Colforsin/pharmacology , Cycloheximide/pharmacology , Cytokines/genetics , DNA, Viral/genetics , Enhancer Elements, Genetic , Extracellular Space/metabolism , HIV-1/physiology , Humans , Indoles/pharmacology , Maleimides/pharmacology , Protein Synthesis Inhibitors/pharmacology , Pyrrolidines/pharmacology , Thiocarbamates/pharmacology , Transcription, Genetic/radiation effects , Viral Proteins/biosynthesis , Virus Replication/radiation effects
11.
Stem Cells ; 16 Suppl 2: 91-5, 1998.
Article in English | MEDLINE | ID: mdl-11012181

ABSTRACT

Targeted gene disruption of two distinct lineage-restricted hematopoietic transcription factors has provided useful insights into the transcriptional control of platelet production. Absence of either the basic leucine-zipper protein NF-E2 or of the zinc-finger protein GATA-1 in vivo results in severe thrombocytopenia secondary to distinct patterns of arrested megakaryocyte cytoplasmic maturation; in addition, megakaryocyte-selective loss of GATA-1 expression leads to dysregulated proliferation of progenitor cells. The ultrastructure of the defective megakaryocytes suggests that absence of the respective transcription factors impairs biogenesis of platelet-specific granules and proper development and organization of demarcation membranes. In particular, transcriptional targets of NF-E2 may be implicated in the very final stages of megakaryocyte differentiation, which involve the organization and release of platelets. Preliminary characterization of genes that are downregulated in NF-E2-/- megakaryocytes is in progress and is likely to lead to mechanistic insights into thrombocytopoiesis.


Subject(s)
Cell Differentiation/genetics , Cell Lineage/genetics , Megakaryocytes/metabolism , Transcription Factors/deficiency , Transcription Factors/genetics , Animals , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Erythroid-Specific DNA-Binding Factors , GATA1 Transcription Factor , Megakaryocytes/cytology , Mice , Mice, Knockout , Mutagenesis, Site-Directed/physiology , NF-E2 Transcription Factor , NF-E2 Transcription Factor, p45 Subunit
12.
J Biol Chem ; 272(35): 21774-83, 1997 Aug 29.
Article in English | MEDLINE | ID: mdl-9268307

ABSTRACT

Stimulation of highly purified primary T lymphocytes through CD2 and CD28 adhesion molecules induces a long-term proliferation, dependent on persistent autocrine secretion of interleukin 2 (IL-2), high and prolonged expression of inducible CD25/IL-2 receptor alpha chain (IL-2Ralpha), and secretion of growth factors such as the granulocyte-macrophage colony-stimulating factor (GM-CSF). CD28 costimulation appears to activate cytokine gene expression through conserved kappaB-related CD28 response (CD28RE) or cytokine 1 (CK-1) elements in addition to canonical NF-kappaB-binding sites. In this report, we assess: 1) the evolution of the expression, over an 8-day time period, of the Rel/NF-kappaB family of proteins in costimulated versus TcR/CD3-stimulated primary T cells; 2) the impact of changes on the in vitro occupancy of GM-CSF kappaB and CK-1, as well as IL-2Ralpha kappaB sites; and 3) the differential regulation of newly synthesized p65 and c-Rel by IkappaB proteins. We show that CD2+CD28 stimulation specifically induces, at maximal T cell proliferation phase, sustained nuclear overexpression of NFKB2 p52 and c-Rel subunits which might rely on long-lasting processing of p100 precursor for p52 and increased neosynthesis of c-Rel. This up-regulation correlates with sustained occupancy of GM-CSF kappaB and CK-1 elements by both proteins. Conversely, these subunits do not appear to bind to the IL-2Ralpha kappaB site. Costimulation, but not TcR/CD3 stimulation, appears supported by sustained down-regulation of both IkappaBalpha and -beta regulators. Furthermore, contrary to p65, c-Rel appears to display little affinity for p105, p100 and IkappaBalpha regulators.


Subject(s)
CD28 Antigens/metabolism , NF-kappa B/metabolism , Proto-Oncogene Proteins/metabolism , Transcription Factors , Cytokines/metabolism , DNA/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Humans , NF-kappa B p50 Subunit , NF-kappa B p52 Subunit , Protein Conformation , Receptors, Interleukin-2/metabolism , Time Factors , Transcription Factor RelA , Transcription Factor RelB
13.
Leukemia ; 11(5): 644-50, 1997 May.
Article in English | MEDLINE | ID: mdl-9180286

ABSTRACT

Pericentric inversion of chromosome 16, translocation (16;16) and del(16q), resulting in a chimerical fusion of CBFbeta and MYH11 genes, are typically seen in the M4Eo French-American-British (FAB) classification subset of acute myelogenous leukemia (AML). In this study, we analyzed 70 cases of acute non-lymphoblastic leukemia, mainly of the M4 or M5 type. We report the very unusual presence of the t(16;16) and CBFbeta/MYH11 fusion transcript in an M7 patient. Ten M4Eo and four non-M4Eo patients presented an inv(16), t(16;16) or CBFbeta/MYH11 fusion transcript. In most cases, the common 'A-type' CBFbeta/MYH11 fusion transcript was detected. In addition to the eight different breakpoints and the three alternative splicing variants already described, evidence of a new CBFbeta/MYH11 fusion transcript was found which involves a 785-bp deletion of MYH11. Moreover, two patients had an unusual transcript, to our knowledge only observed once. Only one patient had abnormal eosinophilic differentiation without chromosome 16 cytogenetic abnormalities or detectable CBFbeta/MYH11 fusion. Conversely, only one patient presented CBFbeta/MYH11 fusion without abnormal eosinophilic differentiation. Altogether, our data suggest a correlation between the CBFbeta/MYH11 fusion transcript and characteristic abnormal eosinophilic differentiation, whatever the FAB subtype or the percentage of abnormal eosinophils


Subject(s)
Chromosome Inversion , Chromosomes, Human, Pair 16 , Leukemia, Myelomonocytic, Acute/genetics , Oncogene Proteins, Fusion/biosynthesis , Translocation, Genetic , Adult , Aged , Base Sequence , Bone Marrow/pathology , Chromosome Deletion , Chromosome Mapping , DNA Primers , Female , Humans , Leukemia, Myelomonocytic, Acute/blood , Leukemia, Myelomonocytic, Acute/classification , Leukemia, Myelomonocytic, Acute/pathology , Male , Middle Aged , Molecular Sequence Data , Polymerase Chain Reaction , Recombinant Fusion Proteins/biosynthesis , Transcription, Genetic
15.
Eur J Biochem ; 244(2): 568-74, 1997 Mar 01.
Article in English | MEDLINE | ID: mdl-9119025

ABSTRACT

Cell-to-cell contact between peripheral blood lymphocytes and transfected human colonic carcinoma cell line HT29 activates transcription of the long terminal repeats (LTR) of human immunodeficiency virus. HIV-1 LTR transcription is controlled by a complex array of virus-encoded and cellular proteins. Using various constructs expressing a lacZ reporter gene under the control of the intact or three deleted forms of HIV-1 LTR, we obtained evidence that the kappaB regulatory elements located in the U3 region are involved in cell-to-cell activation of HIV-1 LTR. Cell-to-cell contact activates in vitro binding of the nuclear factor kappaB (NF-kappaB) p50/p65 heterodimer to an HIV-1 kappaB oligonucleotide. Cell-to-cell contact activation of NF-kappaB was only partially inhibited by 100 microM pyrrolidine dithiocarbamate and was not correlated with a significant decrease of cellular inhibitor kappaB alpha. NF-kappaB nuclear activation was not detectable before 1 h after cell contact and was dependent on protein synthesis.


Subject(s)
HIV Long Terminal Repeat , HIV-1/genetics , Base Sequence , Binding Sites/genetics , Cell Adhesion , Cell Communication , Cell Line , DNA, Viral/genetics , Gene Expression Regulation, Viral , Genes, Reporter , HIV Enhancer , Humans , Lac Operon , Lymphocytes , NF-kappa B/genetics , Transfection
16.
Mol Cell Biol ; 16(12): 6829-40, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8943338

ABSTRACT

The interleukin 2 receptor alpha-chain (IL-2R alpha) gene is a key regulator of lymphocyte proliferation. IL-2R alpha is rapidly and potently induced in T cells in response to mitogenic stimuli. Interleukin 2 (IL-2) stimulates IL-2R alpha. transcription, thereby amplifying expression of its own high-affinity receptor. IL-2R alpha transcription is at least in part controlled by two positive regulatory regions, PRRI and PRRII. PRRI is an inducible proximal enhancer, located between nucleotides -276 and -244, which contains NF-kappaB and SRE/CArG motifs. PRRII is a T-cell-specific enhancer, located between nucleotides -137 and -64, which binds the T-cell-specific Ets protein Elf-1 and HMG-I(Y) proteins. However, none of these proximal regions account for the induction of IL-2R alpha transcription by IL-2. To find new regulatory regions of the IL-2R alpha gene, 8.5 kb of the 5' end noncoding sequence of the IL-2R alpha gene have been sequenced. We identified an 86-nucleotide fragment that is 90% identical to the recently characterized murine IL-2-responsive element (mIL-2rE). This putative human IL-2rE, designated PRRIII, confers IL-2 responsiveness on a heterologous promoter. PRRIII contains a Stat protein binding site that overlaps with an EBS motif (GASd/EBSd). These are essential for IL-2 inducibility of PRRIII/CAT reporter constructs. IL-2 induced the binding of Stat5a and b proteins to the human GASd element. To confirm the physiological relevance of these findings, we carried out in vivo footprinting experiments which showed that stimulation of IL-2R alpha expression correlated with occupancy of the GASd element. Our data demonstrate a major role of the GASd/EBSd element in IL-2R alpha regulation and suggest that the T-cell-specific Elf-1 factor can serve as a transcriptional repressor.


Subject(s)
DNA-Binding Proteins/genetics , Enhancer Elements, Genetic/genetics , Gene Expression Regulation , Interleukin-2/metabolism , Milk Proteins , Proteins/genetics , Receptors, Interleukin-2/genetics , T-Lymphocytes/metabolism , Trans-Activators/genetics , Animals , Base Sequence , Cell Line , Ephrin-A2 , Humans , Mice , Molecular Sequence Data , Receptors, Interleukin-2/metabolism , STAT5 Transcription Factor , Tumor Suppressor Proteins
17.
AIDS Res Hum Retroviruses ; 12(16): 1519-27, 1996 Nov 01.
Article in English | MEDLINE | ID: mdl-8911577

ABSTRACT

Transcription of human immunodeficiency virus type 1 (HIV-1) is regulated by multiple cis-acting regulatory elements located in the viral long terminal repeats (LTRs). HIV-1 LTR enhancer is activated by a variety of heterologous viral, chemical, and physical agents. Studies have demonstrated that irradiation by X-rays induces transcription under the control of the HIV-1 LTR and that ionizing radiations activate DNA binding of the nuclear transcription factor NF-kappa B. Using various constructs expressing a reporter gene under the control of complete or deleted LTRs of HIV-1, we evidenced that a sequence located in the U3 region was involved in X-ray activation of the HIV-1 LTR in the human colonic carcinoma cell line HT29. The cis-acting element conferring X-ray responsiveness is indistinguishable from HIV NF-kappa B tandem repeat binding sites (HIV-1, kappa B). The present work has examined the effects of X-irradiation on the NF-kappa B transcription factor. Furthermore, we characterized the subunit composition of the two major nuclear NF-kappa B complexes that bind HIV-1 kappa B after X-ray irradiation.


Subject(s)
HIV Long Terminal Repeat/radiation effects , HIV-1/radiation effects , NF-kappa B/radiation effects , Antioxidants/pharmacology , Cell Nucleus/metabolism , Colonic Neoplasms , Dose-Response Relationship, Radiation , Gene Expression Regulation , HIV Long Terminal Repeat/genetics , Humans , NF-kappa B/metabolism , Proline/analogs & derivatives , Proline/pharmacology , Thiocarbamates/pharmacology , Transfection , Tumor Cells, Cultured
18.
Cell Mol Biol (Noisy-le-grand) ; 42(6): 811-23, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8891348

ABSTRACT

Recent studies have demonstrated that the activation of HIV-1 provirus and of the Long Terminal Repeat of HIV-1 (HIV-1-LTR) may occur upon cell-to-cell contact between peripheral blood lymphocytes (PBLs) and infected or transfected (HT29) human colonic carcinoma cells. Using transient or stable transfections, we ascertained that the HIV-1 LTR was up-regulated by cell-to-cell contact in various cell lines. The degree of cell-to-cell contact responsiveness was cell type dependent. In contrast, in transient transfection, the HIV-1-LTR was strongly induced by Tat expression in all cell types tested. This indicates that there are differences in the induction mechanism for these two stimuli, even though Tat protein has been previously reported to induce cell adhesion. Except for the PBLs/transfected cells interactions, the results also demonstrate that the cell-to-cell contact-induced HIV-1-LTR activation was highest after contact between autologous as compared to heterologous cells. Previous experiments have shown that, in HT29 cells, cell-to-cell contact activation of the HIV-1-LTR involved the NF-kappa B tandem binding site and activates DNA binding of the nuclear factor NF-kappa B. In this work, we show that in a stably transfected cell line, the principal enhancer element was also involved in the HIV-1-LTR cell-to-cell contact activation. On the other hand, in the HT29 cell line, the NF-kappa B binding appeared to involve the RGD motif of the cell-binding domain, which indicates that a post-integrin receptor event could be implicated.


Subject(s)
Cell Communication/genetics , HIV Long Terminal Repeat/genetics , Cells, Cultured , Colonic Neoplasms/genetics , Colonic Neoplasms/pathology , Humans , Lymphocytes/pathology , Transcriptional Activation , Transfection , Tumor Cells, Cultured
19.
EMBO J ; 14(20): 5060-72, 1995 Oct 16.
Article in English | MEDLINE | ID: mdl-7588634

ABSTRACT

IL-2R alpha transcription is developmentally restricted to T cells and physiologically dependent on specific stimuli such as antigen recognition. To analyse the mechanisms used to activate IL-2R alpha transcription as well as those used to block it in non-expressing cells, we determined the protein-DNA interactions at the IL-2R alpha locus in three different cell types using the DMS/LMPCR genomic footprinting method. CD25/IL-2R alpha can be efficiently induced in primary human T cells since approximately 100% express this gene when receiving an appropriate combination of mitogenic stimuli. To understand why IL-2R alpha is not expressed in other haematopoietic cell types, we analysed BJAB B lymphoma cells which do not express the IL-2R alpha gene and contain constitutively active nuclear NF-kappa B. Primary fibroblasts from embryo and adult skin were selected to examine the mechanisms that may be used to keep the IL-2R alpha gene inactive in non-haematopoietic cells. The three main results are: (i) the stable in vivo occupancy of IL-2R alpha kappa B element in resting T cells, most probably by constitutive NF-kappa B p50 homodimer that could impair SRF binding to the flanking SRE/CArG box; (ii) its inducible occupancy by NF-kappa B p50-p65 associated with the binding of an SRE/CArG box DNA-binding factor upon mitogenic stimulation; and (iii) a correlation between the precommitment of T cells to activation and the presence of stable preassembled protein-DNA complexes in contrast with the bare IL-2R alpha locus in non-T cells.


Subject(s)
Gene Expression Regulation , NF-kappa B/metabolism , Receptors, Interleukin-2/genetics , T-Lymphocytes/metabolism , Transcription, Genetic , Antineoplastic Agents/pharmacology , Base Sequence , Biological Transport , Cell Compartmentation , Cell Differentiation , DNA Footprinting , DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic , Humans , Lymphocyte Activation , Models, Genetic , Molecular Sequence Data , NF-kappa B p50 Subunit , Nuclear Proteins/metabolism , Proline/analogs & derivatives , Proline/pharmacology , Promoter Regions, Genetic , Protein Binding , Serum Response Factor , Thiocarbamates/pharmacology , Tumor Cells, Cultured
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