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H Beug

Publications and source records attributed to H Beug.

At least 109 records · Page 6Linked to original sources
PubMed1990

Receptor-mediated endocytosis of transferrin-polycation conjugates: an efficient way to introduce DNA into hematopoietic cells.

Most current gene transfer methods function satisfactorily in specialized systems involving established cell lines but are often not applicable with nonadherent, primary hematopoietic cells, which are notoriously difficult to transfect. To approach this problem, we have investigated an alternative method of gene transfer, "transferrinfection," in which DNA complexed to transferrin-polycation conjugates is introduced into cells by receptor-mediated endocytosis [Wagner, E., Zenke, M., Cotten, M., Beug, H. & Birnstiel, M. L. (1990) Proc. Natl. Acad. Sci. USA 87, 3410-3414]. We show here that transferrin-polylysine and transferrin-protamine, when complexed to plasmid DNA containing a luciferase reporter gene, is efficiently bound and moved into avian erythroblasts by endocytosis. Successful transfer and expression of the luciferase reporter gene depends on specific interaction of the transferrin-polylysine-DNA complex with the transferrin receptor and occurs in a significant fraction (greater than 95%) of the cells. Gene transfer efficiency by transferrinfection is lower than with an optimized DEAE-dextran transfection method but reaches similar efficiencies when the cells are treated with chloroquine. Because the procedure in the absence of chloroquine is completely nontoxic to cells, a constant expression level of transferred genes may be maintained by repeated additions of transferrin-polylysine-DNA complex. In addition, the usefulness of transferrinfection for gene transfer into primary hematopoietic cells is demonstrated.

Animals↗
PubMed1990

Transferrin-polycation-mediated introduction of DNA into human leukemic cells: stimulation by agents that affect the survival of transfected DNA or modulate transferrin receptor levels.

We have subverted a receptor-mediated endocytosis event to transport genes into human leukemic cells. By coupling the natural iron-delivery protein transferrin to the DNA-binding polycations polylysine or protamine, we have created protein conjugates that bind nucleic acids and carry them into the cell during the normal transferrin cycle [Wagner, E., Zenke, M., Cotten, M., Beug, H. & Birnstiel, M. L. (1990) Proc. Natl. Acad. Sci. USA 87, 3410-3414]. We demonstrate here that this procedure is useful for a human leukemic cell line. We enhanced the rate of gene delivery by (i) increasing the transferrin receptor density through treatment of the cells with the cell-permeable iron chelator desferrioxamine, (ii) interfering with the synthesis of heme with succinyl acetone treatment, or (iii) stimulating the degradation of heme with cobalt chloride treatment. Consistent with gene delivery as an endocytosis event, we show that the subsequent expression in K-562 cells of a gene included in the transported DNA depends upon the cellular presence of the lysosomotropic agent chloroquine. By contrast, monensin blocks "transferrinfection," as does incubation of the cells at 18 degrees C.

Chloroquine↗
PubMed1990

Transferrin-polycation conjugates as carriers for DNA uptake into cells.

We have developed a high-efficiency nucleic acid delivery system that uses receptor-mediated endocytosis to carry DNA macromolecules into cells. We accomplished this by conjugating the iron-transport protein transferrin to polycations that bind nucleic acids. Human transferrin, as well as the chicken homologue conalbumin, has been covalently linked to the small DNA-binding protein protamine or to polylysines of various sizes through a disulfide linkage. These modified transferrin molecules maintain their ability to bind their cognate receptor and to mediate efficient iron transport into the cell. The transferrin-polycation molecules form electrophoretically stable complexes with double-stranded DNA, single-stranded DNA, and modified RNA molecules independent of nucleic acid size (from short oligonucleotides to DNA of 21 kilobase pairs). When complexes of transferrin-polycation and a bacterial plasmid DNA containing the gene for Photinus pyralis luciferase are supplied to eukaryotic cells, high-level expression of the luciferase gene occurs, demonstrating transferrin receptor-mediated endocytosis and expression of the imported DNA. We refer to this delivery system as "transferrinfection."

Animals↗
PubMed1990

Activity and tissue-specific expression of the transcription factor NF-E1 multigene family.

NF-E1, a DNA-binding protein that recognizes the general consensus motif WGATAR, is the first tissue-specific factor to be identified in erythroid cells. Using a probe from the murine GF-1 (NF-E1) cDNA clone, we isolated three homologous chicken cDNAs: One of these corresponds to an mRNA (NF-E1a) that is abundantly and exclusively expressed in erythroid cells; a second mRNA (NF-E1b) is also expressed in all developmental stages of erythroid cells but is additionally found in a limited subset of other chicken tissues; mRNA representative of a third gene (NF-E1c) is expressed only in definitive (adult) erythrocytes within the red cell lineage but is also abundantly expressed in T lymphocytes and brain. All NF-E1 proteins are highly conserved within the DNA-binding domain and bind to the consensus motif with similar affinities in vitro; they are also all stimulatory trans-acting factors in vivo. The factors differ quantitatively in their ability to trans-activate reporter genes in which the number and position of cognate binding sites is varied relative to the transcriptional initiation site. These data suggest that the NF-E1 consensus motif directs a broader and more complicated array of developmental transcriptional regulatory processes than has been assumed and that NF-E1c may play a unique regulatory role in the developing chicken brain and in T lymphocytes.

Amino Acid Sequence↗
PubMed1990

Phosphorylation of the v-erbA protein is required for its function as an oncogene.

The v-erbA oncogene of avian erythroblastosis virus (AEV) encodes a ligand-independent mutated version of the chicken c-erbA alpha-encoded thyroid hormone receptor. The v-erbA gene product, a 75-kD gag/v-erbA fusion protein, is phosphorylated on Ser-16/17 of its v-erbA-encoded domain, and phosphorylation at this site is increased in vivo after activation of either the PKA or PKC signal transduction pathways. To test the hypothesis that phosphorylation of Ser-16/17 regulates gag/v-erbA protein function, mutant proteins in which Ser-16/17 had been changed to alanine or threonine residues were analyzed for their ability to inhibit erythroid differentiation of ts v-erbB or ts v-sea-transformed erythroblasts at nonpermissive temperature. Conversion of Ser-16/17 into alanine, although not affecting nuclear localization or DNA binding of the gag/erbA protein, prevented phosphorylation of the v-erbA-encoded domain of the protein both in unstimulated cells or after stimulation by PKA and PKC activators. The nonphosphorylatable AA-gag/v-erbA protein proved unable to inhibit temperature-induced differentiation of ts v-erbB and ts v-sea-transformed erythroblasts and to block expression of the erythrocyte-specific genes band 3 and carbonic anhydrase II. Back mutation of these alanine residues to serine resulted in the recovery of both normal phosphorylation levels and wild-type biological activity. In contrast, substitution of Ser-16/17 for threonine, which preserved phosphorylation in unstimulated cells but not PKA- and PKC-enhanced phosphorylation, resulted in a partially active gag/v-erbA protein. These results, together with the fact that the protein kinase inhibitor H7 resulted in both a dose-dependent inhibition of gag/v-erbA protein phosphorylation and the induction of terminal differentiation of AEV-transformed erythroblasts show that phosphorylation of gag/v-erbA protein is required for full biological activity. These results support the hypothesis that phosphorylation of the gag/v-erbA protein is important for transcriptional repression of at least some of its target genes in erythroid cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Ectopic expression of the erythrocyte band 3 anion exchange protein, using a new avian retrovirus vector.

A retrovirus vector was constructed from the genome of avian erythroblastosis virus ES4. The v-erbA sequences of avian erythroblastosis virus were replaced by those coding for neomycin phosphotransferase, creating a gag-neo fusion protein which provides G418 resistance as a selectable marker. The v-erbB sequences following the splice acceptor were replaced by a cloning linker allowing insertion of foreign genes. The vector has been tested in conjunction with several helper viruses for the transmission of G418 resistance, titer, stability, transcription, and the transduction and expression of foreign genes in both chicken embryo fibroblasts and the QT6 quail cell line. The results show that the vector is capable of producing high titers of Neor virus from stably integrated proviruses. These proviruses express a balanced ratio of genome length to spliced transcripts which are efficiently translated into protein. Using the Escherichia coli beta-galactosidase gene cloned into the vector as a test construct, expression of enzyme activity could be detected in 90 to 95% of transfected target cells and in 80 to 85% of subsequently infected cells. In addition, a cDNA encoding the avian erythrocyte band 3 anion exchange protein has been expressed from the vector in both chicken embryo fibroblasts and QT6 cells and appears to function as an active, plasma membrane-based anion transporter. The ectopic expression of band 3 protein provides a visual marker for vector function in these cells.

Alpharetrovirus↗
PubMed1990

Requirement for the C-terminal domain of the v-erbA oncogene protein for biological function and transcriptional repression.

In contrast to the normal thyroid hormone receptor, the v-erbA product fails to bind hormone due to mutations in the C-terminal ligand binding domain and thus appears to represent a hormone-independent, oncogenic transcription factor. Therefore, we asked whether or not the C-terminal domain of v-erbA is required for its biological activity and putative transcriptional control functions by analysing mutants with altered C-termini. A v-erbA protein truncated in the C-terminal domain lacked detectable biological activity in transformed erythroblasts and its transcriptional repression function with respect to the band 3 gene was abolished. The protein displayed a nuclear location and could still bind to DNA, indicating that the N-terminal region retained DNA-binding activity but was insufficient to produce characteristic v-erbA changes in erythroblasts. Another biologically defective v-erbA variant with a small frameshift towards the extreme C-terminus also failed to repress band 3, indicating a requirement for a specific C-terminal structure in repression. However, this mutant retained partial biological activity, stimulating erythroblasts to grow at a higher rate than cells containing a completely inactive, deleted v-erbA gene. The results demonstrate that the mutated hormone-binding domain, in addition to the DNA-binding region, is critical for v-erbA biological and transcriptional control functions.

Amino Acid Sequence↗
PubMed1990

The v-erb A oncogene causes repression of erythrocyte-specific genes and an immature, aberrant differentiation phenotype in normal erythroid progenitors.

We have compared the effects of the v-erb A oncogene on proliferation and differentiation of normal erythroid progenitors with those of tyrosine kinase oncogenes, e.g. v-sea. For this, a v-erb A retrovirus containing the neomycin resistance gene as a selectable marker or, alternatively, a v-erb A-ts v-sea retrovirus were used to infect normal bone marrow cells. V-erb A induced the outgrowth of immature, erythropoietin(EPO)-dependent erythroid cells from infected bone marrow which ceased to proliferate and disintegrated after 9 to 18 divisions. In contrast, ts-v-sea erythroblasts grew for the expected 25 to 40 population doublings in the absence of EPO. Transcription of the erythrocyte genes carbonic anhydrase II and erythrocyte anion transporter was significantly inhibited in v-erb A infected erythroblasts, indicating that v-erb A alone was sufficient for the repression of the erythrocyte-specific genes observed in AEV-transformed leukemic cells. A detailed analysis of the differentiation phenotype induced by v-erb A in erythroblasts (in the presence or absence of a temperature-inactivated ts sea oncogene) indicates that v-erb A-erythroblasts express a partially mature, aberrant phenotype characterized by the coexpression of mature and immature differentiation antigens. This phenotype clearly differs from that induced by tyrosine kinase oncogenes in erythroid cells.

Animals↗
PubMed1989

The mutated, myeloid cell-specific growth factor receptor v-fms transforms avian erythroid but not myeloid cells.

In avian hematopoietic cells, transformation by tyrosine kinase oncogenes is restricted to the erythroid lineage. To study the mechanism of this striking target cell specificity, we constructed an avian retrovirus correctly expressing the feline v-fms protein, an oncogenic version of the myeloid-specific CSF-1 receptor. Similar to other tyrosine kinase oncogenes (erbB, src, fps, sea), the v-fms oncogene induced progenitor cells to self-renew in an erythropoietin-independent manner. Spontaneous differentiation of the transformed cells was arrested by v-erbA. v-fms failed to induce transformation of myeloid cells but caused myeloid cells transformed by the v-myb oncogene, as well as normal macrophages, to proliferate independent of cMGF (chicken myelomonocytic growth factor). Unlike the other tyrosine kinases, v-fms did not induce cMGF secretion in myb-myeloblasts, suggesting a nonautocrine mechanism of growth factor independence.

Alpharetrovirus↗
PubMed1988

Hematopoietic growth factor glycosylation. Multiple forms of chicken myelomonocytic growth factor.

The production of chicken myelomonocytic growth factor (cMGF) can be rapidly induced by bacterial lipopolysaccharide from the macrophage cell line HD11. Immunoprecipitation analysis of lipopolysaccharide-induced HD11 cells labeled with various radioactive precursors showed the secretion of a variety of cMGF forms. The precursor-product relationships of the different cMGF forms were studied by pulse-chase experiments, by long-term metabolic labeling in the presence or absence of glycosylation- and oligosaccharide-processing inhibitors, as well as by glycosidase treatment of immunoprecipitates. Our results show that the half-time for intracellular processing/secretion is less than 10 min, making cMGF one of the most rapidly processed proteins. The different forms of the factor are generated from a 24-kDa polypeptide precursor by co- and post-translational acquisition of one or two N-linked oligosaccharides and by O-linked glycosylation. In addition, a fraction of cMGF is modified by long chain, chondroitinase-sensitive, sulfated glycans. This modification is tunicamycin-sensitive, suggesting that the sulfated glycans are attached to N-linked rather than to O-linked oligosaccharides.

Animals↗
PubMed1988

Characterization of the hormone-binding domain of the chicken c-erbA/thyroid hormone receptor protein.

To identify and characterize the hormone-binding domain of the thyroid hormone receptor, we analyzed the ligand-binding capacities of proteins representing chimeras between the normal receptor and P75gag-v-erbA, the retrovirus-encoded form deficient in binding ligand. Our results show that several mutations present in the carboxy-terminal half of P75gag-v-erbA co-operate in abolishing hormone binding, and that the ligand-binding domain resides in a position analogous to that of steroid receptors. Furthermore, a point mutation that is located between the putative DNA and ligand-binding domains of P75gag-v-erbA and that renders it biologically inactive fails to affect hormone binding by the c-erbA protein. These results suggest that the mutation changed the ability of P75gag-v-erbA to affect transcription since it also had no effect on DNA binding. Our data also suggest that hormone-independent activity of P75gag-v-erbA provided a selective advantage to the avian erythroblastosis virus during the original selection for a highly oncogenic strain of the virus.

Animals↗
PubMed1988

Truncation of the human EGF receptor leads to differential transforming potentials in primary avian fibroblasts and erythroblasts.

The transforming capacity of the normal and mutant human EGF receptor (EGFR) was investigated in primary chicken cells. In fibroblasts, both N- and C-terminal truncations resulted in a weak, additive oncogenic activity. However, not even double truncations caused a v-erbB-like phenotype. Upon EGF-binding, on the other hand, both normal and C-terminally truncated EGFRs resembled v-erbB in their fibroblast transforming potential. In erythroblasts, N-terminal truncation was sufficient to induce constitutive self-renewal, which was enhanced by deletion of 32 C-terminal amino acids but abolished by a larger truncation of 202 amino acids. In contrast to the normal EGFR, the receptor lacking 32 C-terminal amino acids resembled v-erbB in conferring erythropoietin independence for spontaneous differentiation to the transformed erythroblasts. Our results indicate that the C-terminal domain of the EGFR is non-essential in fibroblast transformation, but seems to be crucial for both self renewal induction and specificity of receptor function in erythroblasts.

Animals↗
PubMed1988

Temperature-sensitive v-sea transformed erythroblasts: a model system to study gene expression during erythroid differentiation.

The isolation and characterization of a temperature-sensitive mutant (ts1 S13) of the avian erythroblastosis virus, S13, is described. The temperature-sensitive lesion in ts1 S13 was identified as affecting the tyrosine kinase activity but not the plasma membrane localization of the ts1 S13 v-sea gene product. Erythroblasts transformed by ts1 S13 can be induced to synchronously differentiate into erythrocytes in an erythropoietin (EPO)-dependent fashion. Analysis of erythrocyte-specific gene expression in ts1 S13 erythroblasts reveals that the transformed, self-renewing erythroblasts obtained at permissive temperature already express all erythrocyte genes tested for, although at a low level. Upon differentiation induction, expression of erythrocyte-specific genes is not coordinately regulated but rather involves complex regulatory mechanisms that appear to be specific for the individual genes.

Alpharetrovirus↗
PubMed1988

Abnormal glycosylation of the env-sea oncogene product inhibits its proteolytic cleavage and blocks its transforming ability.

Cells transformed by the avian erythroblastosis virus S13 contain three proteins derived from the v-sea oncogene, gp155, gp70 (a cleavage product of gp155), and p38. It is not clear whether only one or all three of these proteins are required for transformation by S13. S13 transformed erythroblasts and fibroblasts revert to a normal morphology in the presence of the alpha glucosidase-1 inhibitor castanospermine, whereas cells transformed by the v-src or v-erbB oncogenes are unaffected by this drug. Treatment with castanospermine does not alter the tyrosine kinase autophosphorylation activity of any of the v-sea products, and the synthesis and processing of p38 is unaffected. Castanospermine modifies the structure of the carbohydrate chains of gp155 such that the glucose residues are retained, thereby inhibiting complex chain formation. Analysis of revertant S13 transformed cells shows that the proteolytic cleavage of the modified form of gp155 is inhibited, resulting in a very low yield of a modified form of gp70. There is no detectable effect of castanospermine on the transport of v-sea gene products to the cell surface. However, due to the inhibition of proteolytic cleavage, the modified form of gp155 is now the major v-sea encoded protein expressed on the cell surface. Thus it appears that the cell surface expression of a v-sea encoded protein with tyrosine kinase autophosphorylating activity is insufficient for cell transformation.

Alkaloids↗
PubMed1988

A point mutation in the DNA binding domain of the v-myb oncogene of E26 virus confers temperature sensitivity for transformation of myelomonocytic cells.

We have molecularly cloned a mutant of the v-myb, ets-containing E26 avian leukemia virus which is temperature sensitive for the transformation of myeloid cells. Cells infected with this mutant, ts21E26, are immature at 37 degrees C and can be induced to differentiate into resting, macrophage-like cells when shifted to 42 degrees C. The sequence of ts21E26 reveals a single relevant nucleotide alteration resulting in a threonine to arginine change in the highly conserved, putative DNA binding v-myb portion of the p135gag-myb-ets protein. Surprisingly, a ts21E26 viral construct in which the v-ets gene domain was deleted was only weakly temperature sensitive, although temperature sensitivity was largely restored in another v-ets deletion mutant whose 3' terminal sequences were replaced with those from the AMV v-myb gene. These results suggest that the temperature sensitive lesion in v-myb of ts21E26 alters the DNA binding capacity of p135 at 42 degrees C and that the primary structure of the C-terminus of this protein has an influence on the activity of sequences that are further upstream.

Animals↗
PubMed1987

v-myb dominance over v-myc in doubly transformed chick myelomonocytic cells.

Chick myelomonocytic cells transformed by the v-myc oncogene resemble mature macrophages; those transformed by v-myb or v-myb,ets exhibit an immature phenotype. We have analyzed whether these oncogenes are capable of altering the differentiation phenotype of transformed cells by introducing both v-myc plus either v-myb or v-myb,ets into the same cells. Surprisingly, the doubly transformed cells were found to be essentially indistinguishable from cells transformed by v-myb or v-myb,ets alone even when they expressed a high level of v-myc protein. These results demonstrate that v-myb is dominant over v-myc and that, while v-myc induces cell proliferation without affecting differentiation, v-myb induces in the same target cells both proliferation and a block or reversal of differentiation.

Animals↗