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D G Tenen

Publications and source records attributed to D G Tenen.

At least 73 records · Page 4Linked to original sources
PubMed1994

Identification of a region which directs the monocytic activity of the colony-stimulating factor 1 (macrophage colony-stimulating factor) receptor promoter and binds PEBP2/CBF (AML1).

The receptor for the macrophage colony-stimulating factor (or colony-stimulating factor 1 [CSF-1]) is expressed from different promoters in monocytic cells and placental trophoblasts. We have demonstrated that the monocyte-specific expression of the CSF-1 receptor is regulated at the level of transcription by a tissue-specific promoter whose activity is stimulated by the monocyte/B-cell-specific transcription factor PU.1 (D.-E. Zhang, C.J. Hetherington, H.-M. Chen, and D.G. Tenen, Mol. Cell. Biol. 14:373-381, 1994). Here we report that the tissue specificity of this promoter is also mediated by sequences in a region II (bp -88 to -59), which lies 10 bp upstream from the PU.1-binding site. When analyzed by DNase footprinting, region II was protected preferentially in monocytic cells. Electrophoretic mobility shift assays confirmed that region II interacts specifically with nuclear proteins from monocytic cells. Two gel shift complexes (Mono A and Mono B) were formed with separate sequence elements within this region. Competition and supershift experiments indicate that Mono B contains a member of the polyomavirus enhancer-binding protein 2/core-binding factor (PEBP2/CBF) family, which includes the AML1 gene product, while Mono A is a distinct complex preferentially expressed in monocytic cells. Promoter constructs with mutations in these sequence elements were no longer expressed specifically in monocytes. Furthermore, multimerized region II sequence elements enhanced the activity of a heterologous thymidine kinase promoter in monocytic cells but not other cell types tested. These results indicate that the monocyte/B-cell-specific transcription factor PU.1 and the Mono A and Mono B protein complexes act in concert to regulate monocyte-specific transcription of the CSF-1 receptor.

Animals↗
PubMed1994

Transcriptional regulation of the human eosinophil peroxidase genes: characterization of a peroxidase promoter.

The molecular basis for commitment of progenitors to the eosinophil lineage and mechanisms by which eosinophil-specific genes are expressed and regulated during differentiation is unknown. Expression of eosinophil peroxidase (EPO) is restricted to the eosinophil lineage. To understand the mechanisms involved in transcriptional regulation of EPO gene expression, we cloned the region of the EPO gene upstream of the transcriptional start site and analyzed the cis-acting elements required for EPO promoter activity in an eosinophil-inducible leukemic cell line, HL60-C15. The -1.5 kb EPO-pXP2 promoter construct reproducibly expressed > 120-fold more luciferase activity than did promoterless pXP2, and a 12-fold decrease in promoter activity was obtained when sequences between -122 and -45 bp were deleted. To further characterize regulatory sequences important for promoter activity, we performed linker-scanning analysis on the -122 to -45 bp region and identified a number of positively and negatively acting elements in the promoter.

Eosinophil Peroxidase↗
PubMed1993

Human eosinophil Charcot-Leyden crystal protein: cloning and characterization of a lysophospholipase gene promoter.

The Charcot-Leyden crystal (CLC) protein is a lysophospholipase expressed exclusively by eosinophils and basophils. During eosinophilic differentiation of eosinophil-committed cell lines, CLC steady state mRNA levels increase significantly. This increased expression is transcriptionally regulated during butyrate induction of an eosinophilic subline (C15) of the promyelocytic leukemia cell line HL-60, as shown by nuclear run-on assays. The transcriptional start site of the CLC gene was identified 43 bp upstream of the 5' end of the longest available cDNA sequence. The gene encoding CLC protein was cloned from a chromosome 19-specific library and a fragment overlapping the transcriptional start site was isolated and sequenced. Plasmid constructs (in the pXP2 luciferase expression vector) containing 411 and 292 bp of genomic sequence upstream of the CLC transcriptional start site directed reporter gene expression in transient transfections of HL-60-C15 cells, as well as other myeloid (U937) and nonmyeloid (HeLa and RPMI 8402) cell lines. However, the differential expression of the two CLC promoter constructs in these cell lines suggests that the -292 to -411 bp region of the promoter may confer some specificity for expression in the eosinophil lineage. The CLC promoter sequence contains two consensus GATA binding sites, a purine-rich sequence that presents potential binding sites for PU.1, a member of the ets family of genes, as well as sequences described in other myeloid-specific promoters. This is the first demonstration of a functional eosinophil promoter that could serve as a model for identifying DNA elements and trans-activating factors that regulate gene expression during the commitment and differentiation of the eosinophil lineage.

Amino Acid Sequence↗
PubMed1993

The Sp1 transcription factor binds the CD11b promoter specifically in myeloid cells in vivo and is essential for myeloid-specific promoter activity.

The myeloid integrin CD11b is expressed selectively on the surface of mature macrophages, monocytes, neutrophils, and natural killer cells. Lineage-specific expression is controlled at the level of mRNA transcription. Recent isolation of the CD11b promoter shows that 92 base pairs (bp) of 5'-flanking DNA are sufficient to direct myeloid-specific expression of a reporter gene. To characterize regulatory sequences important for promoter activity, we performed linker scanning analysis of the 92-bp CD11b promoter and demonstrate that a sequence at bp -60 is essential for CD11b promoter activity. We show that this sequence binds the transcription factor Sp1 in vitro and in vivo. In vivo the Sp1 site is bound only in myeloid (U937) cells, not in cervical carcinoma (HeLa) cells. In addition, the macrophage transcription factor PU.1 binds the CD11b promoter in vitro and in vivo close to the Sp1 site. We propose a model in which binding of a myeloid-specific factor (PU.1) allows a general factor (Sp1) to bind in a tissue-specific fashion thereby contributing to the myeloid-specific expression of CD11b.

Amino Acid Sequence↗
PubMed1993

The proto-oncogene PU.1 regulates expression of the myeloid-specific CD11b promoter.

The myeloid integrin CD11b is expressed selectively on the surface of mature monocytes, macrophages, granulocytes, and natural killer cells. Tissue-specific and developmentally regulated expression of CD11b is controlled at the level of mRNA transcription, and recent characterization of the human CD11b promoter indicates that the first 92 bp of 5'-flanking DNA are sufficient to direct tissue-specific expression of a reporter gene. Here we show that the sequence AAAAGGAGAAG at base pair -20 of the CD11b promoter binds the proto-oncogene PU.1 in vitro and that mutation of this site significantly reduces the ability of the CD11b promoter to direct expression of a reporter gene in myeloid cells but not in nonmyeloid cells. PU.1 may thus represent a major determinant of the myeloid expression of CD11b.

Antigens, CD↗
PubMed1993

Characterization of a distinct binding site for the prokaryotic chaperone, GroEL, on a human granulocyte ribonuclease.

Although ribonucleases fold into correct tertiary conformation in vitro guided solely by information contained in the primary amino acid sequence (Sela, M., White, F. H., and Anfinsen, C. B. (1957) Science 124, 691-693), it is not clear whether folding of these proteins proceeds unassisted in a complex intracellular environment. We describe here the specific and high affinity binding of groEL, the prokaryotic homolog of the heat shock protein 60 family of molecular chaperones, to recombinant eosinophil cationic protein and eosinophil-derived neurotoxin, two members of the human ribonuclease gene family. We have determined that groEL binds to a unique peptide sequence near the amino terminus of nascent eosinophil cationic protein that includes the first of eight cysteine residues. This binding site functions independently and can confer groEL binding activity on an unrelated carrier protein. GroEL dissociates from the binding site upon addition of ATP and Mg2+; no other cations or cofactors are necessary. These findings suggest the possibility that interaction with a groEL-like molecular chaperone may be a requirement for correct folding and/or translocation of eukaryotic ribonucleases in vivo.

Amino Acid Sequence↗
PubMed1993

Molecular cloning and characterization of human eosinophil Charcot-Leyden crystal protein (lysophospholipase). Similarities to IgE binding proteins and the S-type animal lectin superfamily.

We have isolated and sequenced a 598-bp full length cDNA clone for the human Charcot-Leyden crystal (CLC) protein (eosinophil lysophospholipase), the unique and prominent constituent of human eosinophils and basophils that forms the hexagonal bipyramidal crystals classically observed in tissues and secretions from sites of eosinophil-associated inflammation. A 426-bp open reading frame encoded a 142-amino acid polypeptide with a predicted molecular mass of 16.5 kDa and isoelectric point of 7.28. The deduced amino acid sequence of CLC protein showed 20 to 30% similarity over regions of approximately 100 amino acids with the carboxyl-terminal domains of four IgE-binding proteins, including the 31-kDa human and rat IgE-binding proteins, the 35-kDa mouse carbohydrate binding protein (CBP35), Mac-2, the murine macrophage cell surface protein that is identical to CBP35, and the human homologue of Mac-2. These proteins are members of a superfamily of beta-galactoside binding S-type animal lectins, which includes a group of highly conserved 14-kDa lectins isolated from human lung, heart, placenta, bovine heart, chicken skin, mouse fibroblasts, and the electric organ of the electric eel; CLC protein also showed sequence similarities to these 14-kDa animal lectins, including conservation of 7 of 16 invariant amino acid residues thought to comprise the carbohydrate-binding domain of these proteins, with conservative amino acid changes at others; thus, CLC protein could potentially possess carbohydrate or IgE-binding activities. Northern analyses revealed an approximately 900-bp mRNA species that was present in peripheral blood eosinophils from patients with eosinophilia, basophils from patients with chronic myelogenous leukemia, and in HL-60 cells induced towards eosinophilic differentiation with B cell growth factor-II (IL-5) or granulocytic differentiation with DMSO, but was absent in neutrophils, monocytes, T cells, B cells, or HL-60 cells induced towards monocytic differentiation with vitamin D3. Southern analyses revealed a gene of approximately 5 to 6 kb in length. The cDNA clone and complete amino acid sequence data for CLC protein will facilitate structure-function analyses of its unusual hydrophobic properties, unique propensity for crystallization, lysophospholipase, and potential lectin-like activities.

Amino Acid Sequence↗
PubMed1993

Structural analysis of the CD11b gene and phylogenetic analysis of the alpha-integrin gene family demonstrate remarkable conservation of genomic organization and suggest early diversification during evolution.

CD11b is a member of the beta 2 subfamily of the human leukocyte integrins. Its expression is limited to mature myeloid and NK cells and is up-regulated during the course of granulocytic and monocytic differentiation. The CD11b/CD18 (Mo1) heterodimer promotes adhesion of granulocytes and monocytes to C3bi-coated bacteria and endothelial cells. In an attempt to relate the exon structure to the known functional domains, as well as to identify and study cis-acting elements that are involved in its tissue-specific expression, we have isolated genomic clones encoding CD11b, deduced the exon/intron organization, and determined the transcriptional start site. The CD11b gene spans 55 kb and is encoded by 30 exons. Its structure closely resembles that of CD11c, another of the three leukocyte integrin alpha-chains, and suggests that these two genes arose by a gene duplication event. Furthermore, comparison of the CD11b gene structure with that of platelet glycoprotein IIb and Drosophila PS2 suggest how the human leukocyte integrins evolved and dispersed during the course of evolution.

Amino Acid Sequence↗
PubMed1993

Human immunodeficiency virus-1 infection of the human promyelocytic cell line HL-60: high frequency of low-level infection and effect of subsequent cell differentiation.

As a model system to study the infection of early myeloid cells by human immunodeficiency virus-1 (HIV-1), we have infected the human promyelocytic cell line, HL-60, with a recombinant selectable HIV-1 clone. A fully infected population showed a relatively high frequency of low-level infection, with 40% of subcloned cells being negative by reverse transcriptase and p24 indirect immunofluorescence analysis and displaying only low levels of supernatant p24. The same treatment of a T-lymphoid cell line produced 100% productive infections. HIV-1 infection of HL-60 did not appear to alter the state of differentiation of the cells, as assessed by surface antigen expression, regardless of the level of viral expression. Furthermore, infected cells were able to respond normally to chemical inducers of differentiation. Induction of differentiation towards monocyte/macrophages by phorbol myristate acetate activated the HIV-1 long terminal repeat in a transient transfection system, and there was a corresponding increase in viral production from the infected subclones. Granulocytic differentiation, as stimulated by dimethyl sulfoxide or retinoic acid, had no effect on long terminal repeat activity and did not stimulate viral replication. These data suggest that low-level HIV-1 infections may be established at a relatively high frequency in myeloid precursor cells, and that different pathways of promyelocytic differentiation vary in their ability to stimulate HIV-1 replication.

Antigens, CD↗
PubMed1992

The human CD34 hematopoietic stem cell antigen promoter and a 3' enhancer direct hematopoietic expression in tissue culture.

The human CD34 hematopoietic stem cell antigen is a highly glycosylated type 1 membrane protein of unknown function. CD34 is expressed on 1% to 4% of bone marrow cells, including pluripotent stem cells and committed progenitors of each hematopoietic lineage. CD34 has also been shown to be expressed on the small vessel endothelium of a variety of tissues and on a subset of bone marrow stromal cells. We have chosen to use the human CD34 gene as model to examine the transcription factors and cis-elements required for stem cell/progenitor cell-specific gene regulation. We show here that the CD34 gene is transcriptionally regulated in tissue culture cells. Using a luciferase reporter gene, we have isolated and characterized an active CD34 promoter. A CD34-luciferase construct, containing 4.5 kb of 5' flanking DNA from a CD34 genomic clone, was 30-fold more active in CD34+ tissue culture cells than in HeLa cells. Sequences from the 3' end of the CD34 gene were shown to have enhancing activity in CD34+ T-lymphoblastic RPMI-8402 cells and not in CD34- U937 cells or in nonhematopoietic HeLa cells. We also show that a cytidine-guanosine island in the 5' end of the CD34 gene is heavily methylated in two CD34- hematopoietic cell lines and demethylated in two CD34+ cell lines. Analysis of the CD34 promoter should result in the identification of stem cell/progenitor cell-specific transcription factors and should provide a means to direct the expression of heterologous genes in hematopoietic stem cells and progenitors.

Antigens, CD↗
PubMed1992

Cloning and analysis of the CD18 promoter.

CD18, the common beta chain of the leukocyte integrin adhesion proteins, is expressed exclusively by myeloid cells and lymphocytes. During myeloid differentiation, the increase in CD18 cell surface expression is paralleled by increased CD18 messenger RNA levels. Nuclear run-on studies show that CD18 expression is transcriptionally regulated during 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced HL-60 monocytic differentiation. The CD18 transcriptional start site was defined by primer extension and RNAse protection. The gene encoding CD18 was cloned and a fragment that overlaps the transcriptional start site was isolated. DNA sequence analysis of this promoter fragment identified potential AP-1 elements that may mediate the TPA transcriptional response. The CD18 promoter also contains a putative binding site for PU.1, a leukocyte-specific transcription factor. DNA elements resembling those found in other myeloid and integrin promoters were identified. The CD18 promoter fragment was linked to the luciferase reporter gene, electroporated into the U937 monocytic cell line, and its expression increased after exposure to TPA. Thus, CD18 may serve as a model for identifying the cis elements and trans-acting factors that regulate gene expression during myeloid differentiation.

Antigens, CD↗
PubMed1992

Characterization of the myeloid-specific CD11b promoter.

The CD11b/CD18 heterodimeric surface antigen is expressed exclusively on human monocytes, macrophages, granulocytes, and natural killer cells. During differentiation of myeloid cell lines, CD11b steady state messenger RNA levels increase significantly; we show here that CD11b transcription rates increase commensurately. A 1.7-kb fragment of CD11b 5' flanking sequence directs expression of a reporter gene specifically in myeloid cell lines. Deletion analysis localizes elements directing high levels of tissue-specific reporter gene expression to the 412 bp proximal to the transcriptional start site. This sequence contains two consensus binding sites for Sp1, a GATA motif, and a purine-rich sequence that presents potential binding sites for members of the ets family of genes. Analysis of this promoter should result in the isolation of myeloid-specific transcription factors and the development of methods to direct the myeloid-specific expression of heterologous genes.

Antigens, CD↗
PubMed1992

Molecular cloning of a cDNA encoding CD34, a sialomucin of human hematopoietic stem cells.

CD34 is a 115-kDa transmembrane glycoprotein of unknown function that is expressed on human hematopoietic progenitor cells and the small vessel endothelium of a variety of tissues. We have isolated a CD34 cDNA clone from a KG-1 cell library following three rounds of transient expression in COS cells and enrichment by panning with the anti-CD34 mAb MY10 and BI-3C5. The 5' and 3' ends of the full-length cDNA were subsequently amplified by polymerase chain reaction from KG-1 RNA; the final cDNA clone contained 2615 bp and ended in a poly(A) tail. COS cells transfected with the cDNA clone expressed a surface protein of approximately 110 kDa that was immunoprecipitated by MY10. Southern blot analysis suggested that CD34 is a single copy gene. A 2.7-kb CD34 transcript was observed in the hematopoietic cell lines KG-1, KMT-2, AML-1, RPMI 8402, and MOLT 13 and the endothelial cells BAE and EAhy926, but not in monocytes, resting T cells, or the cell lines Laz 509, HL-60, U937, K562, and HeLa. The cDNA sequence predicts a 40-kDa type I integral membrane protein with nine potential N-linked and numerous potential O-linked glycosylation sites in its extracellular domain. There are two consensus protein kinase C phosphorylation sites and one potential tyrosine kinase phosphorylation site in the cytoplasmic portion of CD34. CD34 has no significant sequence homology to any known protein but has some structural similarities to the heavily glycosylated leukocyte surface molecule CD43.

Amino Acid Sequence↗
PubMed1992

The gene for human eosinophil Charcot-Leyden crystal protein directs expression of lysophospholipase activity and spontaneous crystallization in transiently transfected COS cells.

Expression of the gene encoding human eosinophil lysophospholipase, the Charcot-Leyden crystal (CLC) protein, was studied in transiently transfected COS cells. Recombinant CLC (rCLC) protein expression was demonstrated both by Western blot and radioimmunoassay inhibition analyses of transfected COS cell extracts and by immunofluorescent staining and ultrastructural immunogold analyses of intact cells. The rCLC protein was immunochemically indistinguishable from native eosinophil-derived CLC protein, and each transfected COS cell expressed approximately 11 pg of rCLC protein as determined by radioimmunoassay and assessment of transfection efficiency. Immunofluorescent microscopy and ultrastructural immunogold analyses localized rCLC protein to the nucleus, cytoplasm, and plasma membrane of COS cells. Lysates from transfected COS cells producing CLC protein expressed significant lysophospholipase activity. Furthermore, rCLC protein expressed in COS cells spontaneously formed the distinctive intracytoplasmic and intranuclear hexagonal bipyramidal crystals characteristic of the native eosinophil and basophil-derived protein. Expression of the CLC gene confirms the authenticity of the CLC cDNA, the expression of lysophospholipase activity by this unique eosinophil and basophil constituent, and will facilitate the routine purification of the active enzyme for in vitro and animal model studies of its role (or roles) in eosinophil and basophil associated allergic inflammation and eosinophil-parasite interactions.

Animals↗
PubMed1992

Acute myelogenous leukemia: current treatment and future directions.

Acute myelogenous leukemia (AML), although an uncommon disorder, is a useful prototype for the treatment of malignancies in general. Significant advances have been made in both the understanding and treatment of this disease. In particular, clinically relevant molecular mechanisms of disease in AML are being defined that hold future therapeutic promise. We review the classification and biology of AML and the current treatment controversies in the use of chemotherapy and bone marrow transplantation, and suggest directions for future research.

Acute Disease↗
PubMed1992

Structure of the gene encoding CD34, a human hematopoietic stem cell antigen.

CD34 is a cell surface antigen of unknown function expressed in humans in hematopoietic stem cells, vascular endothelium, and blasts from 30% of patients with acute myeloid and lymphocytic leukemia. To begin to investigate the cis-acting elements required for this tissue-specific expression, the human CD34 locus was isolated and its genomic structure and transcriptional start site were characterized. The human CD34 gene spans 26 kb and has 8 exons, a structure quite similar to that of the murine gene. The start site of CD34 transcription was determined to be 258 bp upstream of the translational start site using RNase protection. These experiments also indicated that the 5' untranslated region has extensive secondary structure. In addition, fluorescence in situ hybridization was used to map the CD34 locus to band 1q32.

Amino Acid Sequence↗
PubMed1991

Cellular myosin heavy chain in human leukocytes: isolation of 5' cDNA clones, characterization of the protein, chromosomal localization, and upregulation during myeloid differentiation.

We have isolated 5' cDNA clones encoding a member of the cellular myosin heavy chain gene family from human leukocytes. The predicted amino acid sequence shows 93% identity to a chicken cellular myosin heavy chain, 76% to chicken smooth muscle, and 40% to human sarcomeric myosin heavy chain. The mRNA is expressed as a 7.4- to 7.9-kb doublet in many nonmuscle cells, and is upregulated in myeloid cell lines on induction from a proliferating to a differentiated state. Antisera raised against a peptide made from the predicted amino acid sequence specifically reacts with a 224-Kd polypeptide in leukocyte cell lines, and the protein is also upregulated during the induction of monocytic and granulocytic differentiation in these cells. The gene for this cellular myosin heavy chain maps to chromosome 22, bands q12.3-q13.1, demonstrating that it is not located in the previously described sarcomeric gene clusters on chromosomes 14 and 17. This cellular myosin heavy chain may be a major contractile protein responsible for movement in myeloid cell lines because no mRNA for sarcomeric myosin heavy chain is detected in these cells.

Amino Acid Sequence↗