Biomedical subjects

C Wasylyk

Publications and source records attributed to C Wasylyk.

At least 19 recordsLinked to original sources

PubMed2000

Defect in the p53-Mdm2 autoregulatory loop resulting from inactivation of TAF(II)250 in cell cycle mutant tsBN462 cells.

The cell cycle arrest and proapoptotic functions of p53 are under tight control by Mdm2. After stress activation of p53 by nontranscriptional mechanisms, transcription of the mdm2 gene results in increased synthesis of Mdm2 and down-regulation of p53. Disruption of this autoregulatory loop has profound effects on cell survival and tumorigenesis. We show that a defective p53-Mdm2 autoregulatory loop results from inactivation of a basal transcription factor, TAF(II)250, in tsBN462 cells. We found that Mdm2 expression rescues the temperature-sensitive phenotype of tsBN462 cells, as shown by activation of cell cycle-regulated gene promoters (B-myb, cyclin A, and cdc25C), increased cell growth and DNA synthesis, and inhibition of apoptosis. These effects of Mdm2 are mediated by p53. Exogenous Mdm2 expression apparently complements endogenous Mdm2 synthesis in tsBN462 cells, which is reduced compared to that in the equivalent parental cells with wild-type TAF(II)250, BHK21. Expression of wild-type TAF(II)250 in tsBN462 stimulates and prolongs the synthesis of Mdm2 and rescues the temperature-sensitive phenotype. The TAF(II)250 rescue is blocked by inhibition of Mdm2-p53 interactions. We also show that Mdm2 promoter activation, after transfer to the nonpermissive temperature, is attenuated in cells with mutant TAF(II)250. The temperature-sensitive phenotype apparently results from inefficient inhibition of heat-induced p53 by reduced Mdm2 synthesis due to low mdm2 promoter activity. These results raise the possibility that the p53-Mdm2 autoregulatory loop could guard against transcriptional defects in cells.

Animals↗

PubMed1999

p53 mediated death of cells overexpressing MDM2 by an inhibitor of MDM2 interaction with p53.

The p53 tumour suppressor is frequently inactivated in human tumours. One form of inactivation results from overexpression of MDM2, that normally forms a negative auto-regulatory loop with p53 and inhibits its activity through complex formation. We have investigated whether disrupting the MDM2-p53 complex in cells that overexpress MDM2 is sufficient to trigger p53 mediated cell death. We find that expression of a peptide homologue of p53 that binds to MDM2 leads to increased p53 levels and transcriptional activity. The consequences are increased expression of the downstream effectors MDM2 and p21WAF1/CIP1, inhibition of colony formation, cell cycle arrest and cell death. There is also a decrease in E2F activity, that might have been due to the known physical and functional interactions of MDM2 with E2F1/DP1. However, this decrease is p53 dependent, as are also colony formation, cell cycle arrest and cell death. These results show that a peptide homologue of p53 is sufficient to induce p53 dependent cell death in cells overexpressing MDM2, and support the notion that disruption of the p53-MDM2 complex is a target for the development of therapeutic agents.

Amino Acid Sequence↗

PubMed1997

Conserved mechanisms of Ras regulation of evolutionary related transcription factors, Ets1 and Pointed P2.

Cell transformation by the Ras oncogene is mediated by members of the ets gene family. To analyse the mechanisms of regulation, we have studied activation of several ets factors by Ras expression. We show that expression of Ha-Ras strongly activates the Ets1 p68 and p54 isoforms and Ets2 in F9 EC cells. We have mapped the Ras responsive elements of Ets1 p68 to two domains, RI+II and RIII. Mutation of threonine 82 to alanine in RI+II abolishes both Ras activation and phosphorylation by MAP kinase. Threonine 82 is part of a sequence that is conserved in Drosophila Pointed P2, an ets protein that has been shown both genetically and biochemically to mediate Ras signalling in Drosophila cells. We extend the comparison of these evolutionary related proteins by showing that Pointed P2 is activated by Ras in mammalian cells and mutation of the homologous threonine abolishes activation. Furthermore, we show that Pointed P2 resembles Ets1, in that it has conserved sequences in a similar position adjacent to the ets DNA binding domain that negatively auto-regulates DNA binding. These results go towards showing that the Drosophila Pointed and vertebrate Ets1 are evolutionary related proteins that have remarkably conserved Ras regulatory mechanisms downstream from MAP kinase.

Amino Acid Sequence↗

PubMed1997

Interaction of Ets-1 and the POU-homeodomain protein GHF-1/Pit-1 reconstitutes pituitary-specific gene expression.

The pituitary-specific, POU-homeodomain factor GHF-1/Pit-1 is necessary, but not sufficient, for cell-specific expression of prolactin (PRL), growth hormone (GH), and thyrotropin. Combinatorial interactions of GHF-1 with other factors are likely to be required; however, such factors and their mechanisms of action remain to be elucidated. Here we identify Ets-1 as a factor that functionally and physically interacts with GHF-1 to fully reconstitute proximal PRL promoter activity. In contrast, Ets-2 has no effect, and the alternatively spliced GHF-2/Pit-1beta variant fails to synergize with Ets-1. The Ets-1-GHF-1 synergy requires a composite Ets-1-GHF-1 cis element and is dependent on an Ets-1-specific protein domain. Furthermore, the ancestrally related and GHF-1-dependent GH promoter, which lacks this composite element, does not exhibit this response. Finally, Ets-1, but not Ets-2, binds directly to GHF-1 and GHF-2. These data show that a functional interaction of GHF-1 and Ets-1, acting via a composite DNA element, is required to establish lactotroph-specific PRL gene expression, thus providing a molecular mechanism by which GHF-1 can discriminate between the GH and PRL genes. These results underscore the importance of transcription factors that are distinct from, but interact with, homeobox proteins to establish lineage-specific gene expression.

Animals↗

PubMed1995

Functional interaction of c-Ets-1 and GHF-1/Pit-1 mediates Ras activation of pituitary-specific gene expression: mapping of the essential c-Ets-1 domain.

The mechanism by which activation of common signal transduction pathways can elicit cell-specific responses remains an important question in biology. To elucidate the molecular mechanism by which the Ras signaling pathway activates a cell-type-specific gene, we have used the pituitary-specific rat prolactin (rPRL) promoter as a target of oncogenic Ras and Raf in GH4 rat pituitary cells. Here we show that expression of either c-Ets-1 or the POU homeo-domain transcription factor GHF-1/Pit-1 enhance the Ras/Raf activation of the rPRL promoter and that coexpression of the two transcription factors results in an even greater synergistic Ras response. By contrast, the related GHF-1-dependent rat growth hormone promoter fails to respond to Ras or Raf, indicating that GHF-1 alone is insufficient to mediate the Ras/Raf effect. Using amino-terminal truncations of c-Ets-1, we have mapped the c-Ets-1 region required to mediate the optimal Ras response to a 40-amino-acid segment which contains a putative mitogen-activated protein kinase site. Finally, dominant-negative Ets and GHF constructs block Ras activation of the rPRL promoter, and each blocks the synergistic activation mediated by the other partner protein, further corroborating that a functional interaction between c-Ets-1 and GHF-1 is required for an optimal Ras response. Thus, the functional interaction of a pituitary-specific transcription factor, GHF-1, with a widely expressed nuclear proto-oncogene product, c-Ets-1, provides one important molecular mechanism by which the general Ras signaling cascade can be interpreted in a cell-type-specific manner.

Animals↗

PubMed1994

Reversion of Ras transformed cells by Ets transdominant mutants.

Considerable progress has been made in elucidating the components of the Ras signalling pathway, from both biochemical and genetic investigations. However little is known about the nuclear targets of the pathway, and in particular those that mediate the long-term changes in gene expression resulting from Ras transformation. Ets family members may be involved in these processes since Ras stimulates transcription through ets-DNA binding sites. We show that a mutated Ets protein, delta PU.1, inhibits Ras activation of transcription. Stable expression of delta PU.1 in Ras transformed NIH3T3 fibroblasts reverts the transformed phenotype by many characteristics, including morphology, anchorage independent growth, saturation density, growth in low serum, tumour formation in nude mice and to some extent sensitivity to apoptotic cell death. Similar trans-dominant mutants of c-Ets-1 and c-Ets-2, the most divergent members of the Ets-family to PU.1, also revert Ras transformed cells, as indicated by morphology, anchorage-independent growth, saturation density and doubling time in low serum. Reversion may result from a shared property of the mutants, such as binding to ets motifs in promoters. These results provide evidence for an important role for Ets proteins in Ras transformation.

3T3 Cells↗

PubMed1993

Oncogenic conversion of Ets affects redox regulation in-vivo and in-vitro.

The avian acute leukemia virus E26 encodes a fusion protein between viral Gag and the cellular transcription factors cMyb and cEts1(p68). vEts on its own transforms more mature erythroid cells. We have compared the properties of vEts and cEts1(p68). vEts interacts preferentially with an antibody that recognizes the active conformation of the DNA-binding domain. The DNA-binding activity of vEts is particularly sensitive to incubation conditions for band-shift assays, phosphorylation and modification by sulphydryl-specific reagents. Increased sensitivity is due to loss of a protective function of cEts1 C-terminal sequences. cEts2 has a related C-terminal sequence with a similar role. These results suggest that the vEts DNA-binding domain is more accessible to protein-protein interactions and to regulatory mechanisms. Indeed, vEts DNA binding is preferentially inactivated by oxidizing conditions in-vivo. We suggest that the 'open' conformation of the vEts DNA-binding domain favours interactions with other proteins or DNA and facilitates transformation.

Amino Acid Sequence↗

PubMed1992

A novel modulator domain of Ets transcription factors.

The ets gene family is composed of several oncogenes and codes for transcription factors. The Ets proteins have a similar sequence called the ets domain and bind to the core motif A/CGGAA. We show here that several members of the ets family have different trans-activating properties. The ets domain of Ets-1 is required for DNA binding. Adjacent to this domain there is a novel element that inhibits DNA binding. It appears to alter the structure of the DNA-binding domain before it interacts with DNA. There is a similar sequence in Ets-2 that also inhibits DNA binding. This sequence is absent in alternative splice products of h-Ets-1. PU1, the most distantly related member of the ets gene family, lacks this novel element. It has a distinct DNA-binding specificity that is determined by DNA sequences outside the core motif. These results have important implications for both the oncogenic and normal functions of ets family members.

Amino Acid Sequence↗

PubMed1992

Oncogenic conversion alters the transcriptional properties of ets.

The vEts oncoprotein and its progenitor cEts1(p68) belong to a growing family of transcription factors that are related by the conserved ets domain. We show here that the ets domain and adjacent COOH-terminal amino acids are required for DNA binding by cEts1(p68). vEts differs from cEts1(p68) in both the COOH-terminal sequence and an amino acid substitution in the ets domain. The change in the COOH-terminal sequence markedly decreases its affinity for specific DNA, and the ets domain mutation further diminishes binding. vEts does not trans-activate through the ets (PEA3) motif in vivo. Surprisingly, vEts still efficiently trans-activates the promoters of two genes, stromelysin and collagenase, that are found to be overexpressed in transformed cells. The AP1 motifs of both promoters are required for efficient activation. vEts does not bind to the AP1 motif, even in the presence of cJun and cFos. The DNA-binding domain of Ets1 is required for activation through the AP1 element. Activation is inhibited by the expression of the glucocorticoid and retinoic acid receptors, suggesting that activation by Ets does not involve reversal of negative regulators of AP1. We suggest that activation is by an indirect mechanism involving activation of endogenous genes. Our results show that vEts differs from its progenitor cEts1(p68) in its trans-activating properties. The findings suggest that activation of the Jun and Fos oncoprotein pathway is important for transformation by Ets.

Amino Acid Sequence↗

PubMed1991

The c-Ets oncoprotein activates the stromelysin promoter through the same elements as several non-nuclear oncoproteins.

The c-ets protooncogenes have recently been shown to code for transcription factors that activate the oncogene responsive unit of the polyoma virus enhancer. We show that transcription of the stromelysin gene, which is highly expressed in transformed cells and tumours, is efficiently activated by c-Ets-1 and -2 through two DNA elements. The distal element is a highly conserved palindrome composed of two strong binding sites for c-Ets-1. The proximal element does not bind c-Ets-1, but may be activated indirectly by increased synthesis of c-Jun and c-Fos. Both ets responsive elements mediate activation by the oncoproteins Ha-Ras, v-Src and v-Mos. These results suggest that c-Ets participates in the mechanisms by which stromelysin gene expression is deregulated in transformed cells and tumours.

Animals↗

PubMed1991

Cell-specific regulation of oncogene-responsive sequences of the c-fos promoter.

We have identified oncogene-responsive sequences in the human c-fos promoter that mediate induction of transcription by several nonnuclear oncoproteins and the tumor promoter TPA. These sequences are regulated in a cell-specific manner. (i) In NIH 3T3 cells, the CArG box of the c-fos promoter is sufficient to mediate activation by oncogenes. (ii) In contrast, in HeLa cells, additional flanking sequences are also required, including the outer arm of the serum response element and the FAP site. We also show that the serum response factor, which binds to the CArG box, activates transcription in vivo in NIH 3T3 cells but not in HeLa cells. Finally, we present evidence that the intracellular level of the c-Fos protein could be a major determinant of cell-specific regulation of these oncogene-responsive elements of the c-fos promoter.

Base 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

Oncogene v-jun modulates DNA replication.

Cell transformation leads to alterations in both transcription and DNA replication. Activation of transcription by the expression of a number of transforming oncogenes is mediated by the transcription factor AP1 (Herrlich & Ponta, 1989; Imler & Wasylyk, 1989). AP1 is a composite transcription factor, consisting of members of the jun and fos gene-families. c-jun and c-fos are progenitors of oncogenes, suggestion that an important transcriptional event in cell transformation is altered activity of AP1, which may arise either indirectly by oncogene expression or directly by structural modification of AP1. We report here that the v-jun oncogene and its progenitor c-jun, as fusion proteins with the lex-A-repressor DNA binding domain, can activate DNA replication from the Polyoma virus (Py) origin of replication, linked to the lex-A operator. The transcription-activation region of v-jun is required for activation of replication. When excess v-jun is expressed in the cell, replication is inhibited or 'squelched'. These results suggest that one consequence of deregulated jun activity could be altered DNA replication and that there are similarities in the way v-jun activates replication and transcription.

Base Sequence↗

PubMed1989

PEA3 is a nuclear target for transcription activation by non-nuclear oncogenes.

We have found that the activity of the transcription factor PEA3 is regulated by the expression of non-nuclear oncogenes. This factor, although distinct from PEA1 (AP1), is activated by the same oncogenes (v-src, polyoma (Py) middle T, c-Ha-ras, v-mos, v-raf), by tetradecanoyl phorbol-acetate (TPA) and by serum components. We present evidence that PEA3 and PEA1 co-operate in the response of the polyoma virus (Py) alpha domain to oncogene expression. However, in contrast to PEA1, c-fos does not appear to be necessary for activation of PEA3, suggesting that PEA3 is a fos independent target for regulation of transcription by non-nuclear oncogenes.

Animals↗

PubMed1989

Expression of raf oncogenes activates the PEA1 transcription factor motif.

PEA1 (AP1) motif transcription enhancer activity was stimulated by v-raf and more efficiently by activated c-raf-1 or A-raf than by their normal counterparts, in agreement with a role for PEA1 in transformation by raf. Mutations in the ATP-binding site of v-raf prevented activation, suggesting that phosphorylation is somehow required.

Adenosine Triphosphate↗

PubMed1988

v-jun is a transcriptional activator, but not in all cell-lines.

The recently isolated v-jun oncogene encodes a protein with sequence homology to the transcription factor AP1, as well as a similar DNA binding specificity. We show, by expressing v-jun in F9 embryocarcinoma cells, that v-jun is also a transcriptional activator. However, v-jun expression does not activate transcription in several other cell-lines, suggesting that cell-specific factors are required for v-jun activity.

Animals↗

PubMed1988

A Harvey-ras responsive transcription element is also responsive to a tumour-promoter and to serum.

The ras oncogenes are implicated in the onset of some human tumours, and in cellular proliferation and terminal differentiation. The ras proteins are plasma membrane bound transducers of signals between the outside of the cell and unknown targets in the cell. Identifying these targets and understanding how they are regulated will have a major impact on our understanding of the molecular basis of transformation. We have already shown that c-Ha-ras and the tumor promoter TPA (12-o-tetradecanoyl phorbol-13-acetate) can activate a transcriptional enhancer. We now report the identification of a short sequence in the polyoma virus (Py) enhancer which mediates Ha-ras activation, and show that this sequence (ras responsive element, RRE) also mediates activation by TPA and serum. This responsive element is a specific binding-site for the mouse transcription factor PEA1 (ref. 4 and below) and for the jun oncogene (ref. 5 and M. Karin, personal communication). These results are in keeping with a role for ras protein in signal transduction from outside the cell to a transcription factor in the nucleus, through protein kinase C. The striking similarity between RRE and DNA sequences present in the promoter regions of a number of transformation-related genes suggests that deregulated activation of RRE is a critical event in transformation.

Blood↗

PubMed1988

Transforming but not immortalizing oncogenes activate the transcription factor PEA1.

The transcription factor PEA1 (a homologue of AP1 and c-jun) is highly active in several fibroblast cell lines, compared to its low activity in a myeloma and an embryo-carcinoma (EC) cell line. Serum components are essential to attain these high levels of PEA1 activity in fibroblasts. This serum requirement is abrogated by transformation with the oncogenes c-Ha-ras, v-src and polyoma middle T (Py-MT) but not by immortalization with polyoma large T (Py-LT), v-myc, c-myc or SV40 large T (SV40T). Expression in myeloma cells of the same transforming oncogenes, as well as v-mos and c-fos, activates PEA1, whereas expression of the same immortalizing oncogenes and EIA does not. These results suggest that a common target for transforming oncogenes is PEA1. Serum components have no effect on PEA1 activity in the myeloma and EC cell lines. In contrast, retinoic acid treatment of F9 EC cells augments PEA1 activity. These results suggest that transforming oncogene expression compensates for the absence of cell type-specific factors which are required to activate PEA1. Activation of PEA1 may lead to altered transcription of a set of transformation-related genes.

Antigens, Polyomavirus Transforming↗