Biomedical subjects

D Stehelin

Publications and source records attributed to D Stehelin.

At least 19 recordsLinked to original sources

PubMed1992

Human endothelial cells transfected by SV40 T antigens: characterization and potential use as a source of normal endothelial factors.

A putative role for the vascular endothelium as target for autoantibodies has been suggested in several autoimmune disorders and connective-tissue diseases. However, there are some difficulties linked to the use of cultured endothelial cells (EC) that limit considerably the extensive studies on the nature of endothelial target antigens involved. To overcome this problem, human EC, derived from umbilical veins, were transfected with recombinant plasmid pSV1 which contained the early genes of simian virus SV40. These transfected cells, called EC-pSV1, are able to grow without EC growth supplement and demonstrate a population doubling time of about 50 h. Among the EC properties, EC-pSV1 retain intracellular content of angiotensin-converting enzyme activity, exhibit constitutive production of interleukin 6 and of a growth-promoting activity on early passage EV, express intercellular adhesion molecule 1 (ICAM-1) and its up-regulation by tumor necrosis factor alpha, but have lost the expression of factor VIII-related antigen. Moreover, EC-pSV1 express a 55-kDa antigen found on EC and human platelets, and presumably acting as an antibody target in some cases of non-allergic asthma. However, at the 50-55th generation, morphological changes and altered growth behavior were visible. This work demonstrates that transfection of EC with SV40 T antigens may be of interest, particularly in areas of research including the study of EC targets involved in different human diseases.

Antigens, Polyomavirus Transforming↗

PubMed1992

New chromosome 21 DNA markers isolated by pulsed field gel electrophoresis from an ETS2-containing Down syndrome chromosomal region.

To generate new chromosome 21 markers in a region that is critical for the pathogenesis of Down syndrome (D21S55-MX1), we used pulsed field gel electrophoresis (PFGE) to isolate a 600-kb NruI DNA fragment from the WA17 hybrid cell line, which has retained chromosome 21 as the only human material. This fragment, which contains the oncogene ETS2, was used to construct a partial genomic library. Among the 14 unique sequences that were isolated, 3 were polymorphic markers and contained sequences that are conserved in mammals. Five of these markers mapped on the ETS2-containing NruI fragment and allowed us to define an 800-kb high-resolution PFGE map.

Animals↗

PubMed1992

The ets gene family.

Ets proteins have a conserved DNA-binding domain and regulate transcriptional initiation from a variety of cellular and viral gene promoter and enhancer elements. Some members of the Ets family, Ets-1 and Ets-2, cooperate in transcription with the AP-1 transcription factor, the product of the proto-oncogene families, fos and jun, while others, Elk-1 and SAP-1, form ternary complexes with the serum response factor (SRF). Certain ets gene family members possess transforming activity while others are activated by proviral integration in erythroleukaemias.

Amino Acid Sequence↗

PubMed1992

The c-Ha-ras oncogene induces increased expression of beta-galactoside alpha-2, 6-sialyltransferase in rat fibroblast (FR3T3) cells.

Alteration in cell surface carbohydrates, and in particular cell surface sialylation, have been known to occur during oncogenic transformation. To examine the basis for such changes, we have transformed the rat fibroblast cell line FR3T3 with the oncogenes c-Ha-ras EJ, v-mycOK10, v-src, polyoma virus middle T or the transforming bovine papilloma virus 1 (BPV1), and measured the sialytransferase activities of cellular lysates. We found that, in contrast to all other oncogenes examined, c-Ha-ras induced a striking increase in beta-galactoside alpha-2,6-sialytransferase (Gal alpha-2,6-ST) activity in FR3T3 cells. This increase in Gal alpha-2,6-ST activity resulted in the increased expression of cell surface alpha-2,6-linked sialic acid on cell surface glycoconjugates, as determined by cell staining with fluorescein-labelled Sambucus nigra agglutinin. Immunoprecipitation and immunofluorescence experiments revealed that the increase in Gal alpha-2,6-ST activity was due to an elevation of expression of the enzyme. Moreover, Northern analysis suggested that the increased expression of this enzyme was the result of an increase in the steady-state mRNA level of the Gal alpha-2,6-ST gene. These results support the notion that alterations seen in cell surface glycoconjugates during oncogenic transformation can be the result of altered expression of glycosyltransferases.

Animals↗

PubMed1992

Definition of functional domains in P135gag-myb-ets and p48v-myb proteins required to maintain the response of neuroretina cells to basic fibroblast growth factor.

The v-myb- and v-ets-containing E26 retrovirus induces the proliferation of chicken neuroretina (CNR) cells in minimal medium. Proliferation of E26 CNR cells is strongly stimulated by basic fibroblast growth factor (bFGF). The v-myb-containing avian myeloblastosis virus also induces the proliferation of infected CNR cells stimulated by bFGF. Both E26 CNR and avian myeloblastosis virus CNR cells are able to form colonies in soft agar in the presence of bFGF. This suggests that the v-myb product, a nuclear sequence-specific DNA-binding protein which activates gene expression in transient transfection assays, plays a role in the proliferative response of the infected CNR cells. To determine the structure-function relationships of P135gag-myb-ets and p48v-myb, we have used deletion mutants expressed in retroviral vectors and have analyzed their effect on CNR cell proliferation as well as their effect on the CNR cell response to bFGF. We show that v-ets is not required for bFGF stimulation but increases the proliferation of CNR cells in minimal medium. In the v-myb mutants, the gag sequences derived from the helper virus increase the potency of the myb gene. The carboxyl-terminal domain required for the growth and transformation of myeloid cells and needed for maximal trans-activation in transient DNA transfection assays in fibroblasts was not required for the growth and bFGF response of CNR cells. In contrast, the domain encompassing amino acids 240 to 301 (containing part of the transcriptional activation domain of v-myb) was absolutely required for the response of CNR cells to bFGF and could be functionally replaced by the carboxyl-terminal transcriptional activation domain of the VP16 protein of herpes simplex virus.

Animals↗

PubMed1992

Protein truncation is not required for c-myb proto-oncogene activity in neuroretina cells.

The v-myb oncogene of avian myeloblastosis virus (AMV) differs from its normal cellular counterpart by a truncation at both its amino and carboxyl termini and by a substitution of 11 amino acid residues. We had previously shown that v-myb-containing AMV, in the presence of basic fibroblast growth factor, transformed chicken neuroretina (CNR) cells. To understand the mechanism of c-myb activation, we have tested whether avian retroviruses that express the full-length c-Myb are also active on CNR cells. We have found that c-Myb, like v-Myb, strongly increases the basic fibroblast growth factor response of CNR cells and that these c-myb-expressing cells are able to grow in soft agar in the presence of the growth factor. We have also found that, in contrast to normal or v-myb-expressing AMV-transformed CNR cells, c-Myb-transformed cells express mim-1, a granulocyte-specific gene. However, normal v-Myb- and c-Myb-expressing CNR cells all express the pax-QNR gene, a newly described paired and homeobox-containing gene specifically expressed in the neuroretina. We conclude that, in contrast to what has been described for hematopoietic cells, overexpression of c-Myb is sufficient to activate gene expression and to induce an abnormal behavior of CNR cells.

Avian Myeloblastosis Virus↗

PubMed1992

The various domains of v-myb and v-ets oncogenes of E26 retrovirus contribute differently, but cooperatively, in transformation of hematopoietic lineages.

The genome of the avian leukemia virus E26 is a unique example of association between two transcription factors which appear as a fused composite nuclear oncoprotein, P135gag-myb-ets. Previous studies with E26 have shown that v-myb and v-ets must cooperate to fully transform both erythrocytic and myelomonocytic precursor cells in vivo and in vitro. To analyse further the contribution of the individual domains involved in the transformation of various hematopoietic lineages, we have constructed several mutant viruses expressing a fusion protein with deletions in either v-myb or v-ets. We show here that integrity of the v-ets oncogene is necessary for transformation of the erythrocytic cells but that neither the DNA-binding domain nor the trans-activating domain of v-myb is required for this transformation. The DNA-binding domain of v-ets is necessary to transform myelomonocytic cells. Furthermore, we show that E26 onco-protein also transforms granulocytic cells. The v-ets DNA-binding domain is not necessary to transform them, whereas deleting the v-myb DNA-binding domain strongly reduces transformation of these cells. These data show that the v-myb and v-ets DNA-binding domains provide quite different contributions to the transformation of various hematopoietic lineages by E26.

Animals↗

PubMed1992

Characterization of a paired box- and homeobox-containing quail gene (Pax-QNR) expressed in the neuroretina.

The retina is an integral part of the central nervous system, and consists of two layers, the outer pigmented layer and the inner sensory layer or neuroretina (NR). The NR layer contains several strata of cells (glial and neuronal) derived from proliferating neuroectodermal precursors that differentiate after terminal mitosis. In vitro, NR cells can differentiate not only into neuronal and glial types, but also into pigment and lens cells. Quail (Coturnix coturnix japonica) NR cells (QNR) infected with MC29 transforming retrovirus become pigmented after several passages in vitro. In order to characterize the genes expressed in these pigmented MC29 QNR, a cDNA library was prepared from these cells. After differential screening we have isolated a cDNA clone which identifies an RNA expressed in NR but not in the pigmented layer of the retina. This cDNA encodes a protein related to that of Drosophila, mouse and zebrafish paired box- and homeobox-containing segmentation genes and is called Pax-QNR. The expression of Pax-QNR in the NR is confined to the ganglionic cell layer and to the lower part of the inner nuclear layer containing the amacrine or correlation neurones.

Amino Acid Sequence↗

PubMed1992

c-ets1 proto-oncogene is a transcription factor expressed in endothelial cells during tumor vascularization and other forms of angiogenesis in humans.

The c-ets1 proteins are transcriptional activators expressed within endothelial cells during blood vessel development in chick embryos. The authors show by in situ hybridization that c-ets1 is transcribed in the endothelia during angiogenesis in human embryos, in granulation tissue, and especially during tumor vascularization. c-ets1 mRNAs were also detected in the fibrocytes of tumor stroma and in the spindle cells of Kaposi's sarcomas, regarded as cells of endothelial origin. It has been shown that the c-ets proteins activate transcription through a PEA3 motif that plays a role in the stimulation of transcription of urokinase-type plasminogen-activator (u-PA), stromelysin and collagenase genes. The authors demonstrate in vitro that the angiogenic factor TNF alpha increases transiently the amount of both c-ets1 and u-PA mRNA in confluent human umbilical vein endothelial cells. Therefore, the authors suggest that the c-ets1 proteins might regulate the transcription of the genes coding for matrix-degrading proteases, which are necessary for both angiogenesis and tumor invasion.

Adenocarcinoma↗

PubMed1992

Phylogeny of the p68c-ets-1 amino-terminal transactivating domain reveals some highly conserved structural features.

The chicken c-ets-1 locus gives rise to two distinct transcription factors differing only in their structurally and functionally unrelated N-termini. One of these transcription factors, p54c-ets-1, contains a specific, short (27 amino acids), hydrophilic N-terminus encoded by a single exon, I54, that is widely conserved among vertebrates. The other one, p68c-ets-1, the cellular counterpart of the viral ets oncogene product, differs in the replacement of the I54 by two exons, termed alpha and beta, encoding a larger (71 amino acids), hydrophobic N-terminus which, in contrast to I54, exhibits properties of a transactivating domain. To date the alpha and beta exons have only been found in chicken. Here, we demonstrate the existence of the alpha and beta exons in other avian species (quail and duck) and the existence of the alpha exon in reptiles (turtle). However, none of them could be detected in mammals. Our results strongly suggest that, in contrast to the phylogenetically well-conserved I54 exon, the alpha exon is restricted to reptilian species (birds and 'true' reptiles), whereas the beta exon is detectable so far only in birds. Comparison of their amino acid sequences reveals that the alpha exon and to a much greater extent the beta exon have diverged faster than the I54 exon. In addition, we show that the N- and C-terminal thirds of the alpha exon and the highly hydrophobic nature of the alpha beta-encoded sequence are heavily conserved features and thus likely to be required for function as a transactivating domain in p68c-ets-1 and possibly in the viral P135gag-myb-ets transforming protein.

Amino Acid Sequence↗

PubMed1992

c-ets-1 DNA binding to the PEA3 motif is differentially inhibited by all the mutations found in v-ets.

The proto-oncogene c-ets-1, one of the two cellular sequences transduced by the avian retrovirus E26, encodes for two transcription factors that activate through a purine-rich motif. The v-ets oncogene differs from its cellular progenitor p68c-ets-1 (i) by its fusion to gag- and myb-derived sequences in the E26 P135gag-myb-ets fusion protein, (ii) by two point mutations, and (iii) by the replacement of the 13 C-terminal amino acids present in c-ets-1 by 16 unrelated residues in v-ets. A 35 kDa protein which binds to the purine-rich PEA3 motif in a sequence-specific manner has been obtained by expression in Escherichia coli of the 311 carboxy-terminal amino acids of c-ets-1. Using various v-/c-ets-1 chimeric 35 kDa proteins expressed in bacteria, we have shown that all the mutations found in v-ets, when introduced into this c-ets-1 protein, diminish or even abolish its sequence-specific DNA binding. These results demonstrate that, in addition to the previously defined 85 amino acids located near the carboxy terminus of the c-ets-1 protein (the ETS domain), other sequences are required for sequence-specific DNA binding. In addition, the c-ets-1 35 kDa polypeptide carrying the two point mutations and the viral-specific carboxy terminus, and thus similar to the v-ets-encoded domain of the E26 P135gag-myb-ets, does not bind to the PEA3 motif.

Amino Acid Sequence↗

PubMed1991

Developmental changes of acidic fibroblast growth factor (aFGF) transcription and expression in mouse brain.

In order to increase our knowledge of the in vivo role of acidic fibroblast growth factor (aFGF) in the central nervous system, we have examined aFGF levels during mouse brain development. Using a specific polyclonal antibody raised against aFGF, we measured levels of aFGF-immunoreactive material (IRMaFGF) in extract of total mouse brain taken at different days of development. We found that the level of measurable IRMaFGF remained low and without significant variation during fetal brain development (0.2 ng/mg of extracted proteins). During the first 11 days postnatal (P0 to P11), IRMaFGF increased from 0.5 to 1.5 ng/mg. Between P11 and P14 IRMaFGF levels went up more rapidly, reaching 5 ng/mg. From P30 to adulthood a constant value of 2.5 ng/mg was measured, aFGF content in the different brain extracts was further characterized by its affinity for heparin-Sepharose, its elution at 1 M NaCl from this column and its capacity to induce thymidine incorporation in quiescent fibroblasts. These results were confirmed at the mRNA level. Northern blot analyses of poly A+ mRNA from brains with a specific riboprobe for bovine aFGF, revealed a major 4.5-Kb transcript and a minor 2.7-Kb transcript detectable only in postnatal brains. A similar pattern to that observed for IRMaFGF was seen with these mRNA transcripts, indicating that these aFGFmRNA are translated in the mouse brain. Our results suggest that aFGF may act in the postnatal phases of brain maturation.

Animals↗

PubMed1991

Genomic organization of the human thyroid hormone receptor alpha (c-erbA-1) gene.

The thyroid hormone receptor alpha (THRA or c-erbA-1) gene belongs to a family of genes which encode nuclear receptors for various hydrophobic ligands such as steroids, vitamin D, retinoic acid and thyroid hormones. These receptors are composed of several domains important for hormone-binding, DNA-binding, dimerization and activation of transcription. We show here that the human THRA gene is organized in 10 exons distributed along 27 kbp of genomic DNA on chromosome 17. The position of the introns in human THRA is highly conserved when compared to the chicken gene despite their differing lengths. The N-terminal A/B domain as well as the 5' untranslated region is encoded by two exons. Interestingly, each of the putative zinc fingers of the receptor DNA-binding domain is encoded by one exon and the hormone-binding domain is assembled from three exons. The two last exons of the gene are alternatively spliced to generate two different messenger RNAs. In addition, we confirm that another gene, belonging to the nuclear receptor superfamily, ear-1, overlaps with the 3' region of THRA in an opposite transcriptional orientation.

Amino Acid Sequence↗

PubMed1991

v-myb and v-ets cooperate for the mitogenic stimulation of primary fibroblasts by avian E26 retrovirus.

By using a series of deletion mutants, we have shown that the stimulation of fibroblast growth by E26 requires the cooperation of the two oncogenes, v-myb and v-ets, fused in the nuclear viral product. Of the two DNA-binding domains, only one must be present to promote anchorage-independent growth, whereas that of v-myb is required to allow growth in low serum medium. Furthermore, the v-ets oncogene comprises multifunctional domains.

Animals↗

PubMed1991

An alternatively spliced c-mil/raf mRNA is predominantly expressed in chicken muscular tissues and conserved among vertebrate species.

The chicken c-mil gene produces two mRNA species generated by an alternative splicing mechanism. These two transcripts differ at least by the presence or absence of a 60 nucleotide exon (E7a) localized at the splice junction of c-mil exons 7 and 8. By using RNAase protection assays, we have analysed the pattern of expression of these two mRNA species in several chicken tissues. Here we report that the two c-mil mRNAs are differentially expressed in chicken tissues: the mRNA lacking E7a is detected in all tissues tested, while the mRNA containing E7a is detected only in the skeletal muscle, heart and brain. Sequences homologous to E7a have also been detected in DNAs from quail, mouse and human cells and their sequencing revealed that the alternative E7a is structurally preserved in these species. By PCR analyses performed on RNAs extracted from muscular tissues of these species, we also show that the alternative splicing mechanism described in chicken also occurs in these species.

Amino Acid Sequence↗

PubMed1990

The c-ets proto-oncogenes encode transcription factors that cooperate with c-Fos and c-Jun for transcriptional activation.

Cell transformation by oncogenes leads to changes in gene expression. A key event in this process seems to be activation of the transcription factors AP-1 and PEA 3. Their synergistic activities are required for efficient activation of transcription from different promoters by many different oncogenes, serum growth factors and the tumour promoter TPA. We show here that the products of the ets-1 and -2 proto-oncogenes, whose biological function was previously unknown, are transcription factors that activate transcription through the PEA 3 motif. The p68c-ets-1 protein specifically binds to DNA and contains a transcriptional activation domain. The ets-like gene family therefore seems to encode a new family of transcription factors, apparently unrelated to other transcription factors. The p68c-ets-1 protein cooperates with c-Fos and c-Jun (components of AP-1) for activation of transcription from the oncogene-responsive domain of the polyoma enhancer, indicating that combined activity of all three oncoproteins could be involved in the response of cells to growth stimuli.

Base Sequence↗

PubMed1990

myc products induce the expression of catecholaminergic traits in quail neural crest-derived cells.

The avian myelocytomatosis virus strain MC29 v-myc oncogene transforms a wide panel of avian cells in vitro and either blocks or maintains differentiation, depending on the cell type. In the present work, we have investigated the effect of this oncogene on the differentiation of early embryonic cells, neural crest cells, grown in vitro. We report that the MC29 v-myc gene product induces a strong cellular proliferation of 2-day quail neural crest with the appearance of catecholaminergic traits. Other v-myc as well as the c-myc gene products also trigger this phenotype. Retroviruses carrying some other oncogenes do not elicit this phenotypic expression, although they activate cell multiplication. Thus, our results indicate that myc gene products induce (directly or indirectly) a differentiated phenotype in a subpopulation of neural crest cells.

Animals↗

PubMed1990

The chicken cellular progenitor of the v-ets oncogene, p68c-ets-1, is a nuclear DNA-binding protein not expressed in lymphoid cells of the spleen.

The chicken cellular c-ets-1 locus encodes for two related proteins generated by alternative splicing: the widely expressed p54c-ets-1 protein and the cellular homolog of the v-ets-encoded domain of the E26-transforming protein P135gag-myb-ets, p68c-ets-1 which has been found so far only in the spleen. We have prepared a new site specific antiserum directed against the amino-terminus of p68c-ets-1, which is highly hydrophobic by contrast to the hydrophilic NH2 terminus of p54c-ets-1. This antiserum specifically immunoprecipitated p68c-ets-1 and P135gag-myb-ets only in denaturing and reducing conditions. Despite these biochemical differences at their amino-terminal parts, p68c-ets-1, as p54c-ets-1, is a nuclear protein able to bind to DNA in vitro. Unlike p54c-ets-1 which is expressed at high levels in T- and B-lymphoid cells, p68c-ets-1 is not expressed in lymphoid cells of the spleen. Thus, the two c-ets-1 encoded proteins, although both exhibiting DNA-binding properties in vitro, display differences both in the nature of their specific NH2 termini, and in their level and pattern of expression.

Amino Acid Sequence↗