Biomedical subjects

D Stehelin

Publications and source records attributed to D Stehelin.

At least 55 records · Page 3Linked to original sources

PubMed1993

Evolution of the ets gene family.

Over the past few years a variety of genes have been described whose protein products share similarity with that of the c-ets-1 proto-oncogene, the cellular counterpart of the v-ets oncogene of the avian E26 retrovirus. This so-called "ets family" of transcription factors includes at least a dozen members present in several organisms. We have questioned the common evolutionary origin of these various gene products. By constructing phylogenetical trees with different methods, we show that the ets family is very ancient since the duplication of the various groups of ets related proteins occurred before the Arthropods/Vertebrates split (ca. 500 million years).

Amino Acid Sequence↗

PubMed1993

A new mechanism of oncogenic activation: E26 retroviral v-ets oncogene has inverted the C-terminal end of the transcription factor c-ets-1.

The v-ets-encoded domain in the P135gag-myb-ets transforming protein of the E26 retrovirus differs mainly from its cellular progenitor, p68c-ets-1 by two point mutations and by the replacement of the 13 last C-terminal amino acids present in c-ets-1 by 16 unrelated residues of previously unknown origin in v-ets. Here, we demonstrate that these v-ets C-terminal specific residues are in fact encoded by the opposite strand of the c-ets-1 C-terminus.

Amino Acid Sequence↗

PubMed1993

Characterization of quail Pax-6 (Pax-QNR) proteins expressed in the neuroretina.

After differential screening of a cDNA library constructed from quail neuroretina cells (QNR) infected with the v-myc-containing avian retrovirus MC29, we have isolated a cDNA clone, Pax-QNR, homologous to the murine Pax-6, which is mutated in the autosomal dominant mutation small eye of mice and in the disorder aniridia in humans. Here we report the characterization of the Pax-QNR proteins expressed in the avian neuroretina. From bacterially expressed Pax-QNR peptides, we obtained rabbit antisera directed against different domains of the protein: paired domain (serum 11), domain between the paired domain and homeodomain (serum 12), homeodomain (serum 13), and carboxyl-terminal part (serum 14). Sera 12, 13, and 14 were able to specifically recognize five proteins (48, 46, 43, 33, and 32 kDa) in the neuroretina. In contrast to proteins of 48, 46, and 43 kDa, proteins of 33 and 32 kDa were not recognized by the paired antiserum (serum 11). Paired-less and paired-containing proteins exhibited the same half-life (6 h) and were phosphorylated mostly on serine residues. Immunoprecipitations performed with subcellular fractions of neuroretinas showed that the paired-containing proteins were located in the nucleus, whereas the 33- and 32-kDa proteins were found essentially in the cytoplasmic compartment. However, immunofluorescence experiments performed after transient transfections showed that p46 and p33/32 were also located in vivo into the nucleus. Thus, the Pax-QNR/Pax-6 gene can produce proteins with two DNA-binding domains as well as proteins containing only the DNA-binding homeodomain.

Animals↗

PubMed1993

[Does oncogene c-ets 1 participate in the regulation of tumor angiogenesis?].

The formation of new blood vessels is an essential process in embryonic development and wound healing, for tumor growth and metastasis. In situ hybridization studies have revealed that the protooncogene c-est1 is expressed in endothelial cells at the beginning of blood vessel formation, in normal and pathological conditions. c-ets1 encodes a transcription factor, a protein which binds specifically to DNA and which regulates the transcription of genes containing these specific binding sequences in their promotors. Thus, in vitro experiments suggest that c-ets1 may activate the transcription of genes encoding collagenase 1, stromelysine 1 and urokinase plasminogen activator, proteases involved in extracellular matrix degradation. A working hypothesis is that c-ets1 takes part in regulating angiogenesis by controlling the transcription of these genes whose activity is necessary for the migration of endothelial cells from pre-existing capillaries. This hypothesis is discussed with respect to current experimental evidence and to the complexity of the regulatory network controlling gene transcription and extracellular matrix degradation.

Animals↗

PubMed1993

Involvement of the proto-oncogene c-ets 1 and the urokinase plasminogen activator during mouse implantation and placentation.

Many of the Ets proteins have been shown to be transcription activators. In vitro, Ets 1 proteins are involved in the transcriptional induction of genes such as stromelysin 1, collagenase 1 or urokinase type plasminogen activator, which are proteases responsible for extracellular matrix degradation. In vivo, c-ets 1 is expressed in a wide variety of embryonic tissues in migrating cells, especially in endothelial cells during blood vessel formation. C-ets 1 is also expressed in stromal cells of invasive carcinomas. In the present work, we have investigated the expression of both c-ets 1 and u-PA, a putative target gene of the Ets 1 proteins, within a biological model which includes both embryonic and tumoral aspects. Implantation and placentation of the mouse embryo display migration of the trophoblastic cells, which invade the stroma of the uterine endometrium and trigger the establishment of a new vascular frame. Using in situ hybridization, we show that the overlapping of expression of c-ets 1 and u-PA is restricted to some maternal cell populations from the invasive front and to the endothelial cells of the endometrial vasculature. C-ets 1 is never expressed in trophoblasts. In contrast, u-PA expression in trophoblasts is strong and coincides with the embryo invasive phase. In the embryo proper, c-ets 1 displays a spatio-temporal expression pattern similar to that described in the chick embryo. Until E 10.5, u-PA is expressed neither in embryonic nor in extra-embryonic structures. The respective roles of c-ets 1 and u-PA and their relationship during mammalian placentation are discussed.

Animals↗

PubMed1993

Characterization of a functional promoter for 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 that encode nuclear receptors for various hydrophobic ligands such as steroids, retinoic acid and thyroid hormones. We have previously described the genomic organization of the human THRA gene, which comprises 10 exons distributed along 27 kbp of genomic DNA. We describe here a promoter that initiates THRA transcription. This promoter contains no obvious TATA-like element but is very GC rich and harbors numerous Sp1 sites. It also contains several sites similar to previously described cis-acting sequences including hormone-responsive elements (HREs). When transfected into cultured HeLa cells, it drives the expression of a CAT reporter gene. The activity of this human THRA promoter is enhanced by the synthetic glucocorticoid dexamethasone but seems unaffected by thyroid hormones.

Animals↗

PubMed1993

New human erg isoforms generated by alternative splicing are transcriptional activators.

The erg gene is a member of the ets gene family. ETS proteins have been shown to bind specifically the (GGA-A/T) motif and to transactivate via this consensus sequence. The human erg products exhibit approximately 70% homology with ETS proteins in their DNA-binding domain. We have isolated three erg cDNAs from a human fetal liver library. Two of them are different from the previously described erg-1 and erg-2 cDNAs (Rao et al., Science, 1987, 237, 635-639), in the middle of their coding sequence and in their 5' part where a novel initiation codon is introduced. These isoforms are generated by alternative RNA splicing from a single gene that leads to the inclusion or exclusion of different exon sequences. The three cDNAs expressed by an in vitro transcription-translation system direct the synthesis of proteins of approximately 38, 49 and 55 kDa. These in vitro erg products were tested for their DNA-binding activity by gel mobility-shift assays with different probes containing the ETS-specific binding site. The results indicated that all these erg isoforms are able to bind the ETS binding site in a specific manner. Our data using transient transfection assays indicate that erg protein isoforms function as transcriptional activators.

3T3 Cells↗

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↗