Biomedical subjects

H Beug

Publications and source records attributed to H Beug.

At least 91 records · Page 5Linked to original sources

PubMed1993

Transformation of chicken bone marrow cells by the v-ski oncogene.

The effect of the v-ski oncogene on the transformation of chicken hematopoietic cells was examined. In initial experiments viruses encoding the v-ski oncoprotein did not transform chicken bone marrow cells. However, whereas viruses encoding the ts-v-sea oncoprotein transform solely erythroid cells, viruses encoding both the v-ski and the ts-v-sea oncogenes were found capable of transforming myeloid cells from the monocytic and/or granulocytic lineages in addition to erythroid cells. Analysis of cell clones transformed by the v-ski/ts-v-sea virus identified one clone that no longer expressed the v-sea protein, indicating that this protein was necessary for the initiation but not the maintenance of transformation. Subsequent experiments testing the effects of various growth factors on transformation of bone marrow cells by the v-ski oncogene product alone identified the avian c-kit ligand (stem cell factor; SCF) as being able to co-operate with the v-ski protein to cause transformation of chicken hematopoietic cells of both myeloid and erythroid lineages.

Animals↗

PubMed1993

Mutational analysis of the role of the carboxy-terminal region of the v-erbB protein in erythroid cell transformation.

The v-erbB protein encoded by the avian erythroblastosis virus AEV-H is responsible for the transformation of fibroblasts and erythroblasts by this virus. Deletion of amino acids 961-1102, which lie carboxy terminal to the kinase domain of the v-erbB protein, destroys the ability of the virus to transform erythroid cells without severely affecting fibroblast transformation. This region is termed the E-domain and has been postulated to contain a region important for erythroid cell transformation. In-frame deletions of approximately 23 amino acids were introduced throughout the E-domain in an attempt to locate a specific region that was essential for erythroid cell transformation. Several of the deletions gave rise to a partial transformed phenotype, although no single deletion that completely abolished erythroid cell transformation was found. Interestingly, deletion of amino acids 1031-1055 resulted in a superactivated v-erbB protein that was more active for erythroid cell transformation than the wild-type AEV-H v-erbB protein. This indicates that there is a negative regulatory region located within this region that normally partially suppresses the transforming activity of the v-erbB protein for erythroid cells. These data suggest that the E-domain contains both negative and positive regulatory regions that function in erythroid cells, and complete deletion of the region is necessary to abolish erythroid cell transformation.

Amino Acid Sequence↗

PubMed1992

Activation of an inducible c-FosER fusion protein causes loss of epithelial polarity and triggers epithelial-fibroblastoid cell conversion.

As a novel approach to studying the modulation of the polarized epithelial phenotype, we have expressed c-Fos and c-Myc estrogen receptor fusion proteins (c-FosER and c-MycER) in mammary epithelial cells. The hybrid proteins could be activated by estrogen for defined time periods and after the cells had achieved their fully polarized organization. Activation of c-MycER deregulated proliferation but did not affect epithelial polarity. Short-term activation of c-FosER induced the reversible loss of morphological and functional cell polarity. In contrast, long-term stimulation of c-FosER caused the cells to depolarize irreversibly, to invade collagen gels, and to undergo epithelial-fibroblastoid cell conversion. Our data suggest that Fos proteins are important in modulating the epithelial phenotype both in normal tissue development and in invasive processes.

Animals↗

PubMed1992

Factor-dependent erythroid cell lines derived from mice transplanted with hematopoietic cells expressing the v-src oncogene.

Transplantation of spleen cells from primary reconstituted mice expressing the v-src oncogene to secondary and tertiary irradiated recipients resulted in the emergence of erythroid precursors with a transformed phenotype. When cultured in methyl cellulose, these precursors generated colonies of undifferentiated cells that could be expanded into continuously growing factor-dependent cell lines in liquid culture. All lines tested had a similar phenotype and expressed the v-src oncogene. In addition they responded to factors that regulate normal erythroid development, namely erythropoietin (Epo), interleukin-3 (IL-3), and mast cell growth factor (MGF), the ligand to the c-kit encoded receptor. When cells from one of the lines were maintained in the absence of factor, a "factor independent" subpopulation emerged that appeared to grow in an autocrine fashion. Conditioned medium from these cells stimulated their own growth as well as the growth of broad spectrum of normal precursors. Studies with neutralizing antibodies indicated that the predominant colony-stimulating factor produced by these cells is IL-3.

Animals↗

PubMed1992

Transcriptional repression of band 3 and CAII in v-erbA transformed erythroblasts accounts for an important part of the leukaemic phenotype.

The v-erbA oncogene confers two prominent properties on transformed erythroblasts: a block of spontaneous differentiation and tolerance to wide variations in the pH or ionic strength of culture medium. V-erbA acts as a constitutive repressor of erythrocyte-specific gene transcription, arresting the expression of at least three different erythroid genes: the erythrocyte anion transporter (band 3), carbonic anhydrase II (CAII) and delta-aminolevulinate synthase (ALA-S). To test whether or not the v-erbA induced repression of these genes is causally related to the v-erbA induced leukaemic phenotype, we have reintroduced the genes for band 3 or CAII into transformed erythroblasts via retrovirus vectors. We show here that such erythroblasts, expressing v-erbA, require the same narrow range of medium pH and ion concentration for growth as do transformed erythroblasts lacking v-erbA, i.e. the v-erbA induced tolerance to pH variation was abrogated. The v-erbA induced differentiation block, however, remained unaffected by the re-expression of band 3 and was only slightly affected by the re-expression of CAII. Our experiments show that the two v-erbA-related 'erythroblast transformation parameters' are separable: suppression of band 3 and CAII accounts for one parameter (pH/ion tolerance), while the second parameter (differentiation block) must involve v-erbA regulation of a different set of target genes.

5-Aminolevulinate Synthetase↗

PubMed1992

Hormone-regulated v-rel estrogen receptor fusion protein: reversible induction of cell transformation and cellular gene expression.

We describe the construction of a v-rel estrogen receptor fusion protein (v-relER) which allows the regulation of v-rel oncoprotein activity by hormone. In the presence of estrogen, v-relER readily transformed primary chicken fibroblasts and bone marrow cells in vitro. In both cell types, v-rel-specific transformation was critically dependent on the presence of estrogen or the estrogen agonist 4-hydroxytamoxifen (OHT). Withdrawal of estrogen or application of an estrogen antagonist, ICI164,384 (ICI) caused a reversal of the transformed phenotype. We also demonstrate that the v-relER protein binds to NF-kappa B sites in an estrogen-dependent manner, thereby showing that sequence-specific DNA binding of v-relER is critical for the activation of its transforming capacity. In transient transfection experiments, we failed to demonstrate a clear repressor or activator function of the v-rel moiety in v-relER. However, in v-relER-transformed bone marrow cells, estrogen and OHT induced elevated mRNA levels of two cellular genes whose expression is constitutive and high in v-rel-transformed cells. These results suggest that v-rel might exert part of its activity as an activator of rel-responsive genes.

Animals↗

PubMed1992

Immortalization of conditionally transformed chicken cells: loss of normal p53 expression is an early step that is independent of cell transformation.

Clones of mortal chicken fibroblasts and erythroblasts transformed by temperature-sensitive v-src and v-erb B oncoproteins have been developed into immortal cell lines that retain the conditional transformed phenotype. The expressions of two tumor suppressor genes, the retinoblastoma (Rb) gene and the p53 gene, were investigated during senescence, crisis, and cell line establishment. In temperature-sensitive (ts)-v-erb B erythroblasts and ts-v-src fibroblasts (as well as in v-myc macrophages), loss of p53 mRNA or expression of a mutated p53 gene invariably occurred in the early phase of immortalization. In contrast, expression of the Rb gene was unchanged at all stages of immortalization. Inactivation of the original temperature-sensitive oncogene led to loss of the transformed phenotype in fibroblasts and to differentiation in erythroblasts, even in lines that were immortal and lacked p53. The results demonstrate that the process of immortalization is distinct from cell transformation, probably requiring different mutational events.

Animals↗

PubMed1992

Characterization of early and late endocytic compartments of the transferrin cycle. Transferrin receptor antibody blocks erythroid differentiation by trapping the receptor in the early endosome.

We describe a detailed morphological characterization of the endocytic pathway in differentiating chicken erythroblasts transformed by a temperature-sensitive mutant of avian erythroblastosis virus (AEV). These cells express high levels of transferrin receptors (TfR) when induced to differentiate at 42 degrees C. Biochemical analysis showed that most (approximately 90%) of the internalized 125I-Tf recycled within approximately 30 min while a smaller fraction of 125I-Tf required up to 2 h for recycling. By immunocytochemistry, the bulk of Tf and TfR was localized at the plasma membrane and in tubuloreticular early endosomes. This structure contained coated buds that labelled with an antibody specific for the clathrin light chain. Decreasing amounts of both Tf and TfR were detected in two distal compartments, spherical endosome vesicles resembling multivesicular bodies and the prelysosomal compartment (PLC) enriched in cation-independent mannose 6-phosphate receptor. As shown by fluorescent (FITC-Tf) labelling of living cells, the movement of Tf/TfR complex into these late structures was accompanied by a significant drop in pH from about 6, the value displayed by early endosomes, to values below pH 5.0. Since no detectable 125I-Tf degradation was observed during a 4 h period we believe that the Tf/TfR detected in these late endocytic structures avoids degradation and recycles back to the cell surface. The addition of an anti-TfR monoclonal antibody to the culture medium of these cells blocks their differentiation. Under this condition the antibody-TfR complex was trapped in an early endosome compartment that enlarged to more than twice its normal size. However, this condition did not affect the transport kinetics of horseradish peroxidase from the medium to the PLC.

Alpharetrovirus↗

PubMed1992

cis and trans regulation of tissue-specific transcription.

Analysis of both the cis-regulatory sequences which control globin gene switching as well as the trans-acting factors which bind to these sequences to elicit a differential, developmentally regulated response has lent insight into the general mechanisms responsible for tissue-specific gene regulation. We show here that the chicken adult beta-globin gene promoter sequences are intimately involved in competitive interaction with the beta/epsilon-globin enhancer to regulate differentially epsilon- versus beta-globin gene transcription. Secondly, we show that the family of GATA transcription factors directs gene regulation in a variety of discrete cell types, and describe potential cellular target genes for each member of the GATA factor family, as well as potential mechanisms whereby multiple GATA factors expressed in a single cell might be used to elicit differential transcriptional activities.

Animals↗

PubMed1992

The v-erbA oncogene requires cooperation with tyrosine kinases to arrest erythroid differentiation induced by ligand-activated endogenous c-erbA and retinoic acid receptor.

The v-erbA oncogene, a mutated version of the thyroid hormone receptor alpha (c-erbA/TR-alpha), cooperates with tyrosine kinase oncogenes in erythroblast transformation. Here we show that the ligand-activated, endogenous retinoic acid receptor (RAR-alpha), in cooperation with c-erbA/TR-alpha, efficiently reverses the transforming effect of kinase oncogenes, overcoming oncogene-induced self-renewal by triggering terminal differentiation of the transformed cells into healthy erythrocytes. This differentiation induction was accompanied by up-regulation of erythrocyte gene expression. Similarly, RAR-alpha and over-expressed exogenous c-erbA/TR-alpha efficiently abolished the differentiation arrest caused by v-erbA, while the low levels of endogenous TR-alpha had no effect. In contrast, transformation by v-erbA plus a kinase oncogene was not affected at all by ligand-activated endogenous or over-expressed exogenous TR-alpha and RAR-alpha. These results suggest that oncogene cooperation is required to protect leukemic erythroblasts from differentiation induction via endogenous, nuclear hormone receptors. Endogenous c-erbA/TR-alpha and RAR-alpha apparently cooperated in abolishing erythroblast self-renewal and inducing differentiation, since the respective ligands acted in a synergistic fashion, and overexpressed, non-ligand-bound c-erbA/TR-alpha suppressed endogenous RAR-alpha function in differentiation induction. Genetic evidence is presented that this functional cooperation requires the receptor dimerization domain, suggesting that TR-alpha/RAR-alpha heterodimers play a role in regulation of erythroid differentiation.

Blood Proteins↗

PubMed1992

Modulation of normal erythroid differentiation by the endogenous thyroid hormone and retinoic acid receptors: a possible target for v-erbA oncogene action.

The v-erbA oncogene, a mutated version of the thyroid hormone receptor alpha (c-erbA/TR-alpha), inhibits erythroid differentiation and constitutively represses transcription of certain erythrocyte genes, suggesting a normal function of the proto-oncogene c-erbA in erythropoiesis. Here we demonstrate that the endogenous thyroid hormone receptor alpha (c-erbA/TR-alpha) and the closely related retinoic acid receptor alpha (RAR-alpha) play a role in the regulation of normal erythroid differentiation. Retinoic acid (RA) distinctly modulated the erythroid differentiation program of normal erythroid progenitors and erythroblasts reversibly transformed by a conditional tyrosine kinase oncogene. When added pulsewise to immature cells, differentiation was accelerated while more mature cells underwent premature cell death. Thyroid hormone (T3) alone caused similar but weaker effects. Interestingly, T3 strongly enhanced the action of RA, suggesting cooperative action of the two receptors in modulating erythroid differentiation. Expression of the human RAR-alpha in receptor-negative erythroblasts conferred RA-induced regulation of differentiation to the otherwise unresponsive cells, thus showing that the RAR-alpha is essential for the RA effect. Likewise, enhanced expression of exogenous c-erbA/TR-alpha in erythroblasts rendered them susceptible to modulation of differentiation by T3, suggesting a similar function of both receptors.

Bone Marrow Cells↗

PubMed1992

The leukaemia oncogene v-erbA: a dominant negative version of ligand dependent transcription factors that regulates red cell differentiation?

The v-erbA oncogene of avian erythroblastosis virus alters the growth properties and arrests differentiation of chick erythroid progenitor cells. The v-erbA protein is a mutated, ligand independent version of the c-erbA/T3R alpha chick receptor for T3, a ligand dependent transcriptional regulator. In reconstituted systems using idealized hormone responsive elements, over-expressed v-erbA acts as a dominant repressor of transcription mediated by liganded c-erbA/T3R alpha. This property seems to account for at least part of the phenotype of AEV transformed erythroid cells and for the transcriptional repression of some erythrocyte specific genes. However, v-erbA is likely to interfere with regulatory circuits other than those directly regulated by T3 receptors. Aspects of this hypothesis are discussed in the context of available evidence for the role of T3 and other hormones in erythroid progenitor cells proliferation/differentiation.

Animals↗

PubMed1992

The v-ski oncogene cooperates with the v-sea oncogene in erythroid transformation by blocking erythroid differentiation.

The avian retrovirus oncogene v-ski was analysed for its ability to alter the differentiation program of erythroid cells and to cooperate with tyrosine kinase oncogenes in leukemogenesis. For this, a retrovirus combining v-ski with a temperature-sensitive version of the v-sea oncogene was constructed. In transformed erythroblasts, v-ski disturbed the concerted expression of several erythrocyte genes, leading to an abnormal erythroblast phenotype. Expression levels of hemoglobin and erythrocyte anion transporter (band 3) were elevated, while expression of the erythroid-specific histone H5 was strongly suppressed. v-ski could also be shown to repress or severely retard the temperature-induced erythroid differentiation of v-ski/ts-v-sea-transformed cells. The undifferentiated cells had an abnormal erythroblast or early reticulocyte phenotype characterized by unusually low levels of histone H5. In chicks, the v-ski/ts-v-sea virus displayed enhanced leukemogenicity compared with viruses containing just the single oncogenes. Thus, v-ski cooperates with tyrosine kinase oncogenes in a similar fashion to the v-erbA oncogene, however the pattern of genes affected by these two oncogenes is different.

Animals↗

PubMed1992

Avian erythroblastosis: a model system to study oncogene co-operation in leukemia.

Avian erythroblastosis virus, AEV-ES4, provides an ideal in vitro system to study oncogene co-operation in the development of erythroid leukaemias in chickens. Two oncogenes that use distinct signal transduction pathways have been identified; these act to produce a highly malignant phenotype and are an oncogenic version of a plasma membrane growth factor receptor that regulates haematopoietic progenitor self renewal and a mutated version of a transcription factor that suppresses specific gene expression. These two diverse types of oncogenes act together to generate a more malignant phenotype than would be expected merely from a summation of the individual oncogene effects. The aspects of normal growth control and the regulation of differentiation, which are altered in this system to generate the leukaemic phenotype, include changes in the balance between proliferation versus maturation, altered expression of differentiation genes and changes in growth factor responsiveness. These are exactly those features that are altered in human leukaemias. Thus, the avian leukaemia virus model has taught us important lessons that will help us to understand the basic molecular mechanisms that give rise to human leukaemia.

Alpharetrovirus↗

PubMed1991

Primary structure and expression of a chicken cDNA encoding a protein with zinc-finger motifs.

We report the cloning and sequence analysis of a chicken nearly full-length cDNA clone, cKr1, encoding a protein of 509 amino acids which contains ten 2Cys + 2His-type zinc-finger motifs arranged in two separate sets of five zinc fingers each. The cKr1 transcripts are detected in organs of the adult chicken, predominantly in the brain and lung. At day 4 of embryonic development strong cKr1 hybridization signals are found in the brain and neural tube, in the mesonephros and in the gut, respectively.

Alpharetrovirus↗

PubMed1991

v-erbA overexpression is required to extinguish c-erbA function in erythroid cell differentiation and regulation of the erbA target gene CAII.

The v-erbA oncoprotein represents a retrovirus-transduced oncogenic version of the thyroid hormone (T3/T4) receptor c-erbA (type alpha). It contributes to virus-induced erythroleukemia by efficiently arresting differentiation of red cell progenitors and by suppressing transcription of erythrocyte-specific genes. Here, we show that v-erbA and c-erbA bind directly to sequences within the promoter of the erythrocyte-specific carbonic anhydrase II (CAII), a gene whose transcription is efficiently suppressed by v-erbA. This erbA-binding site confers thyroid hormone responsiveness to a heterologous promoter in transient expression experiments and is a target for efficient down-regulation of CAII transcription by the v-erbA oncoprotein. In stably transformed erythroblasts coexpressing the v-erbA oncoprotein and the c-erbA/T3 receptor at an approximately equimolar ratio, c-erbA activity is dominant over v-erbA. T3 efficiently induced erythroid differentiation in these cells, thus overcoming the v-erbA-mediated differentiation arrest. Likewise, T3 activated CAII transcription as well as transient expression of a T3-responsive reporter gene containing the CAII-specific erbA-binding site. The c-erbA-dependent activation of this CAII reporter construct could only be suppressed by very high amounts of v-erbA. Our results suggest that overexpression of v-erbA is required for its function as an oncoprotein.

Base Sequence↗

PubMed1991

Expression of v-rel in a replication competent virus: transformation and biochemical characterization.

The avian reticuloendotheliosis virus strain T (REV-T) transforms bone marrow cells and may cause phenotypic changes in fibroblasts. Both events are thought to result from expression of the v-rel oncoprotein, a member of the NF-kappa B family of transcription factors. Most REV stocks contain a cytopathic and immunosuppressive helper virus (REV-A) unrelated to standard avian retroviruses, and thus the degree to which v-rel expression alone contributes to the transformed phenotype in bone marrow cells and fibroblasts is complicated by helper virus expression. To gain a more accurate picture of how v-rel contributes to transformation, we have cloned the v-rel gene into a replication-competent avian retrovirus vector (RCAS) and have expressed it in both chick embryo fibroblasts (CEF) and bone marrow cells. Transfection of RCAS-rel into CEF readily produced a partially transformed phenotype, demonstrating that expression of the v-rel protein is sufficient for fibroblast transformation. The RCAS-rel virus also transformed bone marrow cells in vitro, but required culture conditions different from those normally required for transformation by REV-T. The v-rel protein expressed in transformed CEF was biochemically indistinguishable from that expressed in transformed bone marrow cells, being localized to the cytoplasm and the nucleus, and forming a complex with cellular proteins. We also demonstrate that the RCAS-rel-transformed hematopoietic cells exhibited a distinct differentiation phenotype.

Animals↗

PubMed1990

v-erbA oncogene activation entails the loss of hormone-dependent regulator activity of c-erbA.

The v-erbA oncogene, one of the two oncogenes of the avian erythroblastosis virus, efficiently blocks erythroid differentiation and suppresses erythrocyte-specific gene transcription. Here we show that the overexpressed thyroid hormone receptor c-erbA effectively modulates erythroid differentiation and erythrocyte-specific gene expression in a T3-dependent fashion, when introduced into erythroid cells via a retrovirus. In contrast, the endogenous thyroid hormone receptor does not detectably affect erythroid differentiation. The analysis of a series of chimeric v-/c-erbA proteins suggests that the v-erbA oncoprotein has lost one type of thyroid hormone receptor function (regulating erythrocyte gene transcription in response to T3), but constitutively displays another function: it represses transcription in the absence of T3. The region responsible for the loss of hormone-dependent regulator activity of v-erbA has been mapped to the very C-terminus of c-erbA, encompassing a cluster of highly conserved amino acid residues with the potential to form an amphipathic alpha-helix.

Alpharetrovirus↗