Regulatory issues involved in hematopoietic growth factor approval.
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Animals↗
Biomedical subjects
R B Cohen
Publications and source records attributed to R B Cohen.
Concanavalin A-induced granulocyte-macrophage colony-stimulating factor production in a murine T-cell line is posttranscriptionally controlled.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor (HGF) that regulates the proliferation and differentiation of cells of the myeloid lineage. It can be produced by a variety of cells. One of the major sources of GM-CSF is activated T cells, which transiently produce this HGF. We used the EL-4 thymoma cell line as a model system to address the molecular basis for GM-CSF regulation in T cells. Both concanavalin A (ConA) and the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) induce GM-CSF expression in EL-4 cells. However, the biological activity of GM-CSF in the supernatants of the TPA-stimulated cells was higher than that of ConA-stimulated cells. To elucidate this difference in biological activity levels, we examined how ConA regulates GM-CSF gene expression in EL-4 cells and compared it to the better-characterized regulation by TPA. Peak mRNA levels of GM-CSF occur 6 h after stimulation with either of these two agents. GM-CSF mRNA levels after ConA treatment are lower and decrease significantly after 10 h compared to TPA treatment, which causes much higher levels that persist for at least 24 h. Neither agent alters GM-CSF gene transcription. Actinomycin D chase experiments show that ConA increases the GM-CSF mRNA half-life from less than 30 to 90 min, whereas TPA prolongs it to greater than 3 h. These results indicate that GM-CSF mRNA induction by ConA (in common with TPA) is regulated predominantly via RNA stabilization and that the difference in prolongation of the mRNA half-life provides the primary explanation for the lower levels of GM-CSF mRNA induced by ConA compared to TPA.
Animals↗
Identification of sequences within the murine granulocyte-macrophage colony-stimulating factor mRNA 3'-untranslated region that mediate mRNA stabilization induced by mitogen treatment of EL-4 thymoma cells.
Phorbol esters (TPA) and concanavalin A (ConA) are known to induce granulocyte-macrophage colony-stimulating factor (GM-CSF) production in murine thymoma EL-4 cells by mRNA stabilization. The role of the 3'-untranslated region (3'-UTR) in GM-CSF mRNA stabilization induced by TPA and ConA in EL-4 cells was examined by transfection studies using chloramphenicol acetyltransferase (CAT) constructions. The GM-CSF 3'-UTR contains a 63-nucleotide region at its 3' end with repeating ATTTA motifs which is responsible for mRNA degradation in a variety of cell types (Shaw, G., and Kamen, R. (1986) Cell 46, 659-666). We produced constructs containing most of the GM-CSF 3'-UTR (303 nucleotides, pRSV-CATgm) or the 3'-terminal AT-rich region (116 nucleotides, pRSV-CATau) and measured CAT enzyme activity and CAT mRNA after transient transfection into EL-4 and NIH 3T3 cells. Low levels of CAT activity were seen in both cells with either plasmid compared with levels of CAT activity obtained with pRSV-CAT. TPA treatment caused an approximately 10-fold increase in CAT activity and mRNA in EL-4 cells transfected with pRSV-CATgm. No increases were seen in EL-4 cells transfected with pRSV-CATau or pRSV-CAT. No response to TPA was detected in transfected NIH 3T3 cells, indicating that the response to TPA is relatively cell-specific. There was no increase in CAT activity after ConA treatment in EL-4 or NIH 3T3 cells transfected with any of the constructs suggesting that the GM-CSF 3'-UTR lacks elements that can respond alone to ConA. Nuclear run-on and actinomycin D chase experiments in EL-4 cells showed that TPA induces CAT activity via mRNA stabilization. By linker-substitution mutagenesis we show that TPA inducibility depends on a 60-nucleotide region of the 3'-UTR whose 5' end is located 160 nucleotides upstream of the 5' end of the AU-rich region.
Animals↗
IL-1 alpha induces granulocyte-macrophage colony-stimulating factor gene expression in murine B lymphocyte cell lines via mRNA stabilization.
IL-1 has been shown to induce granulocyte-macrophage (GM)-CSF production in several cell types including T lymphocytes, fibroblasts, and endothelial cells. We have previously demonstrated that bacterial LPS activates expression of the GM-CSF gene in murine B lymphocytes. Inasmuch as LPS is also an inducer of IL-1, we asked whether IL-1 could itself induce GM-CSF gene expression in B lymphocytes. The GM-CSF-dependent PT-18 cell line was used to detect GM-CSF biologic activity and a semi-quantitative polymerase chain reaction assay was developed to detect and measure GM-CSF mRNA. IL-1 alpha stimulated GM-CSF production by the murine B cell lines M12.4.1 and TH2.2 but not by the M12 line in a dose- and time-dependent fashion. GM-CSF mRNA expression in IL-1 alpha-treated M12.4.1 cells also increased in a dose- and time-dependent fashion. Nuclear run-on transcription assays revealed constitutive transcription of the GM-CSF gene in untreated M12.4.1 cells that did not change after IL-1 alpha treatment. Actinomycin D chase experiments indicated that IL-1 alpha increased GM-CSF mRNA stability in the M12.4.1 cells. These findings indicate that IL-1 alpha induces GM-CSF gene expression in murine B cell lines and that this event is posttranscriptionally controlled mainly at the level of mRNA stabilization.
Animals↗
Vascular A10 cell membranes contain an endothelin metabolizing neutral endopeptidase.
We have investigated the possible presence of endothelin-metabolizing neutral endopeptidase (NEP, EC 3.4.24.11) on A10 cell membranes using [125I]-ET-1 binding and direct measurements of NEP. NEP activity of A10 cell membranes has been compared to that of solubilized rat kidney brush border membranes (KNEP). Specific [125I]-ET-1 (50 pM) binding (defined with 100 nM ET-1) to A10 cell membranes was increased in a concentration dependent manner by the selective NEP inhibitors thiorphan, phosphoramidon, and SQ 28,603 [(+/-)-N-[2-(mercaptomethyl)-1-oxo-3-phenylpropyl]-beta-alanine] with EC50 values of 9.4, 28.4, and 5.7 nM respectively. At equilibrium (150 min), 70% more specific binding was apparent in the presence of these inhibitors. Phosphoramidon (2 microM) did not alter Bmax values, but it decreased the apparent KD for [125I] ET-1 from 63 (+/- 3) to 27 (+/- 2) pM. Thiorphan, phosphoramidon, and SQ 28,603 inhibited A10 cell NEP activity with IC50 values of 5.3, 36.5, and 6.0 nM respectively, which was similar to values obtained with KNEP (3.6, 22.6, and 3.5 nM). ET-1 inhibited A10 cell NEP, and KNEP with IC50 values of 30 and 21.3 microM respectively. The order of inhibitory potencies: ET-3 greater than ET-1 = ET-2 greater than or equal to sarafotoxin-6b was similar for both systems. These data suggest A10 cell membranes contain a NEP which has similar characteristics to NEP 24.11, and which actively metabolizes [125I]-ET-1.
Alanine↗
The effects of the putative potassium channel activator WAY-120,491 on 86Rb efflux from the rabbit aorta.
WAY-120,491 [(-)-(3S-trans)-2-[3,4-dihydro-3-hydroxy-2,2-dimethyl-6-(trifluoromet hox y)- 2H-1-benzopyran-4-yl]-2,3-dihydro-1H-isoindol-1-one] is a novel antihypertensive agent. We have investigated the effects of this compound on contractile force and 86Rb efflux, using the rabbit aorta, in order to assess its K channel activator properties. K channel blockers and ionic conditions thought to modulate specific K channel types have been used to provide insight into the K channel(s) affected by this compound. WAY-120,491 evoked relaxation of precontracted rabbit aortic rings and increased the rate of 86Rb efflux from strips of rabbit aorta; both effects occurring in a concentration-dependent manner. The WAY-120,491 (1 microM)-induced 86Rb efflux was inhibited by tetraethylammonium (IC50 = 0.38 mM), indicating that the increased efflux was mediated by K channels. Glyburide completely blocked the WAY-120,491 (1 microM)-evoked 86Rb efflux with 50% block occurring at a concentration of 0.48 microM. Glyburide also antagonized the WAY-120,491-induced relaxation of aortic rings. Omission of Ca from the solution bathing the aorta did not inhibit the WAY-120,491 induced 86Rb efflux but rather caused an augmentation of the response. It is concluded that WAY-120,491 may be classified as a K channel opener. Furthermore, the K channel upon which WAY-120,491 acts exhibits some characteristics normally associated with the ATP regulated K channel although the involvement of other K channel types has not been ruled out.
Animals↗
Post-transcriptional regulation of granulocyte-macrophage colony-stimulating factor synthesis in murine T cells.
The granulocyte-macrophage CSF (GM-CSF) gene is known to be controlled at a variety of levels in different cell types. We showed previously that GM-CSF production by lectin or phorbol ester (12-O-tetradecanoyl-phorbol-13-acetate (TPA]-treated T cells was unaffected by cyclosporin A whereas IL-2 and IL-3 expression were. Cyclosporin A is thought to inhibit transcription that suggests that IL-2 and IL-3 are regulated primarily at the transcriptional level while GM-CSF is not. The lack of coordinate gene expression is of particular interest because all three mRNA share the presence of adenosine uridine-rich sequences in the 3' untranslated region and these sequences are believed to act by modulating mRNA stability. We measured the level of GM-CSF mRNA in untreated cells and found it to be extremely low. GM-CSF mRNA levels increased approximately 60-fold within 6 h of TPA-treatment. Nuclear run-on transcription analysis of the same cells showed readily detectable GM-CSF transcription in unstimulated cells that increased less than twofold after TPA treatment. However, IL-2 transcription was insignificant before TPA addition. Actinomycin D chase experiments showed that GM-CSF transcripts in untreated cells have a very short half-life (approximately 45 min) although transcripts in TPA-treated cells have a half-life exceeding 3 h. These findings indicate that GM-CSF production in EL-4 cells treated with TPA is regulated predominantly by modulation of cytoplasmic mRNA half-life.
Animals↗
Increased eIF-2 alpha expression in mitogen-activated primary T lymphocytes.
G0 human T cells synthesize protein at low rates and contain very low levels of eIF-2 alpha mRNA. eIF-2 alpha plays a pivotal role in the earliest regulated steps of translation initiation. We examined eIF-2 alpha gene expression in normal human T cells stimulated with PHA. Nuclear run-on assays indicate low rates of eIF-2 alpha gene transcription in G0 cells and these change 2-fold with PHA treatment. Actinomycin D chase experiments show that the t1/2 of eIF-2 alpha mRNA is similar in G0 and PHA-treated T cells. Analysis of nuclear RNA with probes specific for eIF-2 alpha intron sequences shows that increased eIF-2 alpha expression after PHA treatment is largely due to intranuclear stabilization of the primary transcript. The increase in eIF-2 alpha mRNA does not require new protein synthesis. Hence, expression of this gene appears to be a part of the primary response program of T cells when they are exposed to mitogen.
Cell Nucleus↗
Identification and characterization of a novel transcription factor participating in the expression of eukaryotic initiation factor 2 alpha.
We have recently cloned and characterized the promoter region of the gene encoding eukaryotic initiation factor 2 alpha (eIF-2 alpha) to identify regulatory elements of this housekeeping gene. We compared the location of DNase I-hypersensitive (HS) sites with the distribution of protein-binding sites as revealed by footprint analysis. The eIF-2 alpha promoter contains four upstream DNase I-HS sites extending from -650 to -40 base pairs and a fifth downstream site near the first intron-exon junction. In vitro DNase I footprint analysis shows eight distinct DNA-protein interactions organized into clusters that correspond well with the distribution of the five HS sites. None of the protected regions, however, shares obvious sequence homology with the binding sites of known regulatory factors. To initiate our analysis of factors required for eIF-2 alpha expression, selected a CAP-proximal element shown by in vivo methylation protection analysis to bind a potential regulatory factor. A striking feature of this element is its palindrome sequences and eight-base pair direct repeats. We have purified to near homogeneity a 66-68-kDa protein that binds to this region and have designated it alpha-PAL. The alpha-PAL-binding site extends from -74 to -10. By methylation protection analysis and mobility shift assay, the alpha-PAL-binding site is shown to be two adjacent sites, one with high and one with lower affinity, which bind alpha-PAL in a noncooperative manner. When the high affinity binding site is cloned upstream of the adenovirus 2 core promoter, in vitro transcription is stimulated 2-3-fold. When linked to a CAT reporter gene, activity of the eIF-2 alpha promoter shows an approximate 2-fold dependence on the alpha-PAL element.
Base Sequence↗
A common protein binds to two silencers 5' to the human beta-globin gene.
The temporal sequence of expression of human globin genes during development suggests precise regulation of these genes. Recent studies have characterized a number of DNA sequences within or flanking the human beta-globin gene which are important in its regulation and several proteins which bind to these sequences have been identified. We have found two proteins which bind 5' to the human beta-globin gene. One of these proteins, which we designate BP1, binds to two sequences, one between -550 and -527 bp relative to the cap site, the other between -302 and -294 bp. A second protein, BP2, binds to sequences between -275 and -263 bp. The binding sites for both BP1 and BP2 are in two regions which function as silencers in a transient expression assay using the human erythroleukemia cell line K562. These results and others presented here suggest that BP1 may act as a repressor protein. Negative regulation seems to be an important component of tissue and developmental specific globin gene regulation.
Animals↗
Isolation and characterization of the promoter and flanking regions of the gene encoding the human protein-synthesis-initiation factor 2 alpha.
The promoter region of the gene (eIF-2 alpha) for eukaryotic initiation factor 2 alpha (eIF-2 alpha) was isolated from a human genomic library and its structure was determined by restriction mapping and nucleotide (nt) sequence analysis. The promoter region and twelve in vivo transcriptional start points (tsp) have been identified by endonuclease S1 mapping and their location confirmed by primer-extension analysis, using RNA isolated from human cells. The untranslated leader is 102 to 140 nt long depending upon the tsp, and the 5' region of the mRNA has the potential for forming stable stem-loop structures. The nt sequence of the regions upstream and downstream from the tsp contains neither a 'TATA box' nor a 'CAAT box', but does contain several direct and inverted repeats, as well as palindromic sequences near the tsp. In addition, multiple consensus binding sites for a wide variety of regulatory proteins are present throughout upstream and downstream tsp-flanking regions.
Base Sequence↗
Bacteriostatic effect of tetracycline in a temporary cement.
Temporary crowns with subgingival margins can result in gingival inflammation. This inflammation is the result of plaque accumulating at the restoration-tooth interface and a change in the nature of that plaque to one associated with periodontitis. It was postulated that tetracycline incorporated in a slightly soluble cement would be released in the sulcus and decrease the amount, and improve the quality, of the subgingival microflora. The unknown was whether tetracycline is inactivated by any of the components of the temporary cement Temp Bond. To determine this concern, disks of Temp Bond cement with and without varying concentrations of tetracycline were anaerobically incubated in cultures of Bacteroides gingivalis. Results showed that tetracycline incorporated in Temp Bond at 10% by weight is highly bacteriostatic while retaining typical working properties of the cement.
Bacteriological Techniques↗
Proteins binding to regulatory elements 5' to the human beta-globin gene.
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Binding, Competitive↗
Identification of a downstream sequence and binding protein that regulate adenovirus major late promoter transcription in vitro.
Gel electrophoresis mobility shift and DNase I footprint assays detect a cellular nuclear protein in extracts made from uninfected human cells which binds to a downstream promoter sequence (DPS) in the human adenovirus 2 major late promoter. By DNase I footprint and mutation analyses, we have determined that this new regulatory element extends from positions +146 to +165 (relative to the cap site at position +1). We show by UV cross-linking that a 40-kDa polypeptide specifically binds to this region. Mutations within the DPS which decrease protein binding by 80-90% also cause a 2.5-3-fold decrease in in vitro major late promoter transcription efficiency. Alteration of the template in the 5'-flanking region of the DPS does not affect nuclear protein binding or transcription efficiency. Interestingly, a T----G transversion at position +160 which increases protein binding also impairs promoter activity.
Adenoviruses, Human↗
Method of objective assessment of analgesia in the dog.
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Animals↗
DNA affinity labeling of adenovirus type 2 upstream promoter sequence-binding factors identifies two distinct proteins.
A rapid affinity labeling procedure with enhanced specificity was developed to identify DNA-binding proteins. 32P was first introduced at unique phosphodiester bonds within the DNA recognition sequence. UV light-dependent cross-linking of pyrimidines to amino acid residues in direct contact at the binding site, followed by micrococcal nuclease digestion, resulted in the transfer of 32P to only those specific protein(s) which recognized the binding sequence. This method was applied to the detection and characterization of proteins that bound to the upstream promoter sequence (-50 to -66) of the human adenovirus type 2 major late promoter. We detected two distinct proteins with molecular weights of 45,000 and 116,000 that interacted with this promoter element. The two proteins differed significantly in their chromatographic and cross-linking behaviors.
Adenoviruses, Human↗
The distribution of immunoreactive chromogranins, S-100 protein, and vasoactive intestinal peptide in compound tumors of the adrenal medulla.
Three adrenal medullary tumors that showed admixtures of pheochromocytomatous elements with ganglioneuroma or ganglioneuroblastoma were studied to determine the distribution of immunoreactive chromogranins, S-100 protein, and vasoactive intestinal peptide (VIP). Two tumors consisted of typical pheochromocytoma cells admixed with mature-appearing ganglioneuroma. The third consisted of pheochromocytoma admixed with ganglioneuroblastoma and contained many immature and cytologically atypical cells. In all cases, chromogranin staining was absent or weak in neuronal perikarya and moderate to intense in varicosities of neuronal processes, a finding consistent with the presumed distribution of secretory granules in neurons. Chromogranin staining was also intense in chromaffin cells. Glial cells that stained for S-100 were randomly scattered among chromaffin cells but accumulated in areas with neuronal processes. Weak staining for VIP was present in neuronal cells in one of the two tumors with ganglioneuromatous features. Intense staining for VIP occurred in the third tumor in both neuronal and apparently nonneuronal cells. We conclude that granule distribution and cell-cell interactions for specific cell types in compound tumors tend to mimic those in normal adrenal medulla and sympathetic ganglia. Although immunoreactive VIP was localized exclusively to neurons in one tumor, as in normal tissues, patterns of staining for VIP across tumors are less predictable.
Adrenal Gland Neoplasms↗
Case report 377: Intraosseous lipoma (angiolipoma) of right third rib.
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Adult↗