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Biomedical subjects

M F Shannon

Publications and source records attributed to M F Shannon.

54 records · Page 3Linked to original sources
PubMed1992

Three essential promoter elements mediate tumour necrosis factor and interleukin-1 activation of the granulocyte-colony stimulating factor gene.

Granulocyte-colony stimulating factor (G-CSF) is a haemopoietic growth factor produced by mesenchymal cells but not T lymphocytes after stimulation with specific cytokines or mitogens. A 330 bp promoter fragment of the human G-CSF gene induced reporter gene expression in human embryonic lung fibroblasts in response to tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 beta (IL-1 beta). The same promoter fragment was not active in Jurkat T cells nor did it respond to phorbol ester in either cell type. At least three distinct elements, the CK-1 sequence, a decanucleotide present in haemopoietic growth factor genes, an NF-IL-6 consensus sequence and a consensus octamer sequence, were essential in the G-CSF promoter for TNF-alpha and IL-1 beta response. Mutation of any of these sequences abolished promoter function. In contrast, mutation of two other consensus protein binding sequences, i.e. a Pu-1 site and a CK-2-like sequence, did not eliminate promoter function. Both the CK-1 and octamer sequences acted independently as TNF-alpha and IL-1 beta responsive elements upstream of a heterologous promoter. The response of the octamer sequence and the 330 bp promoter but not the CK-1 sequence was greater with IL-1 beta than TNF-alpha reflecting a similar response of the endogenous gene.

Base Sequence↗
PubMed1992

Human GM-CSF receptor alpha-chain gene is highly polymorphic but not rearranged in AML.

Acute myeloid leukaemia (AML) blast cells express haemopoietic growth factor receptors. However, their presence does not predict response to the cognate ligand in vitro. This suggests that haemopoietic growth factor receptor structure or function may be abnormal in some cases of acute myeloid leukaemia. The granulocyte-macrophage colony-stimulating factor receptor alpha-chain gene (GM-CSF-R) has recently been localised to the pseudoautosomal region of the sex chromosomes. A sex chromosome is lost in 25% of cases of AML FAB subtype M2. The loss of one allele of this gene may have some aetiological significance in AML if the other allele is altered leading to abnormal receptor structure, function or number. In this initial study, we have examined DNA from leukaemic cells of 29 patients with AML, including three with FAB subtype M2 with deletion of an X or Y chromosome for evidence of gross rearrangement of this gene. We report that although the gene is highly polymorphic for a number of restriction enzymes, we have found no evidence of gross rearrangement in AML.

Adolescent↗
PubMed1991

A granulocyte-colony-stimulating factor gene promoter element responsive to inflammatory mediators is functionally distinct from an identical sequence in the granulocyte-macrophage colony-stimulating factor gene.

A number of mesenchymal cells produce hemopoietic growth factors in response to inflammatory mediators in vitro and in vivo. Induced transcription from the hemopoietic growth factor genes is at least partially responsible for their increased expression. We have previously identified a sequence, cytokine (CK)-1, in the granulocyte (G)-CSF gene promoter that responds to TNF-alpha and binds a transcription factor, NF-GMa. We report here that the CK-1 sequence responds in a time- and dose-dependent manner to IL-1 beta and that the mutations which affect NF-GMa binding correlate with decreased transcriptional activity after stimulation with either TNF-alpha or IL-1 beta. The CK-1 sequence also responds to the human T lymphotrophic virus-1 transactivator, tax, so that this promoter element may contribute to the overall response of the G-CSF gene to these various agents. Although NF-GMa binding is seen in a number of cell types, the ability of the G-CSF CK-1 sequence to act as a transcriptional enhancer is specific for fibroblasts and not T cells. Furthermore, we show that an identical sequence in the granulocyte macrophage CSF gene, although apparently binding the same protein in vitro, cannot respond to any of these stimuli in either fibroblasts or T cells. Modification interference experiments, using the CK-1 region in the context of the granulocyte macrophage-CSF and G-CSF genes, indicated that the contact points for NF-GMa differ in each case and suggest that differences in sequences flanking the 10-bp CK-1 region probably leads to an altered DNA:protein conformation, which may explain the differential response of this conserved promoter element.

Base Sequence↗
PubMed1991

Human interleukin-3 mRNA accumulation is controlled at both the transcriptional and posttranscriptional level.

Interleukin-3 (IL-3) is a hematopoietic growth factor that regulates the differentiation of multilineage and committed progenitor cells and the functions of some mature blood cells. The expression of human IL-3 appears to be restricted to stimulated T lymphocytes. We have investigated the kinetics and mechanisms involved in the induction of IL-3 expression in the human T lymphocytic tumor cell line Jurkat. We show that accumulation of IL-3 mRNA is controlled at both the transcriptional and posttranscriptional level. Transcription of the IL-3 gene in these cells appears to be constitutive but no IL-3 mRNA was detected in unstimulated cells, indicating that in resting cells IL-3 mRNA is highly unstable. Treatment with phytohemagglutinin (PHA) induced a small and transient increase in the IL-3 gene transcription rate and led to the production of detectable levels of IL-3 mRNA and protein. Optimal induction of IL-3 expression required a second stimulus. Costimulation of Jurkat cells with both phorbol myristate acetate and PHA caused both a transient increase in IL-3 gene transcription, which is dependent on new protein synthesis, and also a transient increase in mRNA stability.

Cell Line↗
PubMed1990

A novel tumor necrosis factor-responsive transcription factor which recognizes a regulatory element in hemopoietic growth factor genes.

A conserved DNA sequence element, termed cytokine 1 (CK-1), is found in the promoter regions of many hemopoietic growth factor (HGF) genes. Mutational analyses and modification interference experiments show that this sequence specifically binds a nuclear transcription factor, NF-GMa, which is a protein with a molecular mass of 43 kilodaltons. It interacts with different affinities with the CK-1-like sequence from a number of HGF genes, including granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte (G)-CSF, interleukin 3 (IL-3), and IL-5. We show here that the level of NF-GMa binding is induced in embryonic fibroblasts by tumor necrosis factor-alpha (TNF-alpha) treatment and that the CK-1 sequence from the G-CSF gene is a TNF-alpha-responsive enhancer in these cells. The NF-GMa protein is distinct from another TNF-alpha-responsive transcription factor, NF-kappa B, by several criteria. Firstly, several NF-kappa B-binding sites, although having sequence similarity with the CK-1 sequence, cannot compete efficiently for NF-GMa binding to CK-1. Secondly, the CK-1 sequence from both G-CSF and GM-CSF does not respond to phorbol ester treatment as would an NF-kappa B-binding element. These results demonstrate that NF-GMa is a novel transcription factor inducible by TNF-alpha and binds to a common element in HGF gene promoters.

Base Sequence↗
PubMed1989

Synthesis and expression of the gene encoding human interleukin-3.

To perform structure-function studies of human interleukin-3 (hIL-3) we have synthesized a cDNA encompassing the complete coding region of 484 bp. The strategy we employed involved construction of the cDNA in four sections. Each fragment contained six to ten oligodeoxyribonucleotides. Unique restriction sites were engineered to flank the natural sequence for cloning. Naturally occurring restriction sites were placed internally to these, to allow ligation of the four fragments. The gene was cloned into a modified pJL4 vector and expressed in COS cells. Biological assays of supernatants collected from these cells, for both mature cell function and proliferative activity, showed that synthetic hIL-3 had the same activity as that previously determined for recombinant hIL-3.

Animals↗
PubMed1988

Localization of the human G-CSF gene to the region of a breakpoint in the translocation typical of acute promyelocytic leukemia.

The colony-stimulating factors regulate growth, differentiation, and function of blood cells. The effect of granulocyte colony-stimulating factor (G-CSF) on myeloid leukemias is unique among colony-stimulating factors in driving the leukemic cells from a self-renewing malignant state to a mature differentiated phenotype with the concomitant loss of tumorigenicity. This property of G-CSF has led to suggestions that its absence is responsible for lack of differentiation of leukemic cells and that the therapeutic administration of G-CSF could reverse this defect and result in a cure for leukemia. Here we show that the gene coding for human G-CSF is localized to chromosome 17, bands q11.2-21. The translocation of the long arm of chromosome 17 at q12-21 to chromosome 15 is a specific abnormality occurring in a high proportion of, if not all, patients with acute promyelocytic leukemia, a disease characterized by undifferentiated myeloid cells and a dismal prognosis. Abnormalities of the regulation of a specific differentiation factor gene mediated by a specific chromosomal rearrangement may be directly implicated in the pathogenesis of human leukemia.

Chromosome Mapping↗
PubMed1988

Interleukin 4 is at 5q31 and interleukin 6 is at 7p15.

DNA probes to the human interleukin 4 (IL4) and interleukin 6 (IL6) genes have been used for in situ hybridization to normal human chromosomes and Southern blot analysis of a series of mouse-human hybrid cell lines. IL4 maps to 5q31, the same location as IL5 and other haemopoietic growth factor genes. IL6 maps to 7p15. The significance of these locations is discussed.

Chromosome Mapping↗
PubMed1988

Nuclear proteins interacting with the promoter region of the human granulocyte/macrophage colony-stimulating factor gene.

The gene for human granulocyte/macrophage colony-stimulating factor (GM-CSF) is expressed in a tissue-specific as well as an activation-dependent manner. The interaction of nuclear proteins with the promoter region of the GM-CSF gene that is likely to be responsible for this pattern of GM-CSF expression was investigated. We show that nuclear proteins interact with DNA fragments from the GM-CSF promoter in a cell-specific manner. A region spanning two cytokine-specific sequences, cytokine 1 (CK-1, 5' GAGATTCCAC 3') and cytokine 2 (CK-2, 5' TCAGGTA 3') bound two nuclear proteins [nuclear factor (NF)-GMa and NF-GMb] from GM-CSF-expressing cells in gel retardation assays. NF-GMb was inducible with phorbol 12-myristate 13-acetate and accompanied induction of GM-CSF message. NF-GMb was absent in cell lines not producing GM-CSF, some of which had other distinct binding proteins. NF-GMa and NF-GMb eluted from a heparin-Sepharose column at 0.3 and 0.6 M KCl, respectively. We hypothesize that the sequences CK-1 and CK-2 bind specific proteins and regulate GM-CSF transcription.

Base Sequence↗
PubMed1987

Characterization of the six chicken histone H1 proteins and alignment with their respective genes.

Six histone H1 subtypes and histone H5, isolated from chicken erythrocyte nuclei, were visualized on acid/urea polyacrylamide gels. Four of the H1 subtypes have been purified to homogeneity by fast protein liquid chromatography on a strong cation exchange column. The other two subtypes were obtained as enriched fractions from the same fast protein liquid chromatography experiments. Because six chicken H1 genes have been completely sequenced (Coles, L.S., Robins, A. J., Madley, L.K., and Wells, J. R. E. (1987) J. Biol. Chem. 262, 9656-9663), it was possible to assign each of the six H1 proteins to a specific gene after amino acid sequence analysis of peptides derived from the subtypes.

Amino Acid Sequence↗
PubMed1987

Stimulation of proliferation, differentiation, and function of human cells by primate interleukin 3.

Cloned gibbon interleukin 3 (gIL-3) was found to stimulate the proliferation and differentiation of human bone marrow cells to produce day-14 granulocyte, macrophage, granulocyte-macrophage, and eosinophil colonies in semisolid agar. In the presence of normal human plasma, gIL-3 stimulated megakaryocytes. In methylcellulose cultures, it stimulated erythroid colonies in the presence, but not in the absence, of erythropoietin. When mature human leukocytes were used, gIL-3 stimulated the function of purified mature eosinophils as measured by the capacity to kill antibody-coated target cells, to produce superoxide anions, and to phagocytize opsonized yeast particles in a manner similar to recombinant human granulocyte-macrophage colony-stimulating factor. In contrast, gIL-3 did not significantly stimulate any of the neutrophil functions tested, whereas human recombinant granulocyte-macrophage colony-stimulating factor was active in these assays. Among cytokines that are active on human hematopoietic cells, gIL-3 thus has a distinct set of functions and may predict the range of actions of the human molecule.

Animals↗
PubMed1987

Localization of the G-CSF gene on chromosome 17 proximal to the breakpoint in the t(15;17) in acute promyelocytic leukemia.

The human granulocyte-colony stimulating factor gene (G-CSF) is localized at 17q11.2-q21, the region of one of the breakpoints in the 15;17 chromosome translocation specific for acute promyelocytic leukemia (APL). As G-CSF induces differentiation and loss of tumorigenicity in myeloid leukemic cells or cell lines, it was possible that the translocation in APL involved the DNA of the G-CSF coding region or its regulatory region. In situ hybridization to chromosomes with the t(15;17) from patients with the APL translocation using a G-CSF cDNA clone revealed that the coding region of this gene is proximal to the t(15;17) breakpoint on chromosome 17. Southern analysis of DNA from patients with the APL translocation showed no differences in hybridization between normal and leukemic cells. These results indicate that the G-CSF coding sequence is not disrupted by the chromosomal rearrangement characteristic of APL.

Acute Disease↗
PubMed1985

Histone H5 and H1 cross-reacting material is restricted to erythroid cells in chicken.

Monoclonal H5 antibodies and a polyclonal antiserum, raised against the globular domain of chicken H5 (GH5) but which cross-reacts with histone H1(0) from mouse liver, were used to search for H5 or H1 (0)-like proteins in chicken embryo and adult tissue sections by indirect immunofluorescence. Chicken cell lines in culture were examined for H5 protein and H5 mRNA. Histone H5 was detected only in erythroid cells in tissue sections of chicken embryos or adult livers. H5 protein and H5 mRNA were found only in erythroid cells in culture. No cross-reacting proteins were detected in any other tissue or cell line examined.

Animals↗