Biomedical subjects

B Wasylyk

Publications and source records attributed to B Wasylyk.

At least 37 records · Page 2Linked to original sources

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↗

PubMed1999

MDM2 overexpression generates a skin phenotype in both wild type and p53 null mice.

The MDM2 proto-oncogene is overexpressed in human tumours and regulates the activities of the tumour suppressors p53 and pRB. We created mice that overexpress MDM2 under the control of the CMV promoter. These mice did not display an increased tumour incidence, but rather a specific skin phenotype, characterized by desquamation and hyperkeratosis. Transgenic MDM2 was found to be overexpressed in the epidermis, a tissue that normally expresses high levels of MDM2. The phenotype appeared during the first week after birth and then lessened with age, closely following the level of expression of the transgene. MDM2 overexpression was associated with an increase in proliferation in the basal layer, thickening of the epidermis, altered expression of the differentiation markers cytokeratin CK14, CK10 and CK1, and a decrease in the size and the number of granules that contain products of differentiation. Transgenic mice on a p53 null background displayed similar although not identical changes, showing that the effects of MDM2 are to a certain degree p53 independent. The skin is a major site of MDM2 expression in mice, raising the possibility that MDM2 overexpression perturbs the normal pattern of MDM2 expression and inhibits differentiation of the epidermis.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1999

The net repressor is regulated by nuclear export in response to anisomycin, UV, and heat shock.

The ternary complex factors (TCFs) are targets for Ras/mitogen-activated protein kinase signalling pathways. They integrate the transcriptional response at the level of serum response elements in early-response genes, such as the c-fos proto-oncogene. An important aim is to understand the individual roles played by the three TCFs, Net, Elk1, and Sap1a. Net, in contrast to Elk1 and Sap1a, is a strong repressor of transcription. We now show that Net is regulated by nuclear-cytoplasmic shuttling in response to specific signalling pathways. Net is mainly nuclear under both normal and basal serum conditions. Net contains two nuclear localization signals (NLSs); one is located in the Ets domain, and the other corresponds to the D box. Net also has a nuclear export signal (NES) in the conserved Ets DNA binding domain. Net is apparently unique among Ets proteins in that a particular leucine in helix 1, a structural element, generates a NES. Anisomycin, UV, and heat shock induce active nuclear exclusion of Net through a pathway that involves c-Jun N-terminal kinase kinase and is inhibited by leptomycin B. Nuclear exclusion relieves transcriptional repression by Net. The specific induction of nuclear exclusion of Net by particular signalling pathways shows that nuclear-cytoplasmic transport of transcription factors can add to the specificity of the response to signalling cascades.

Amino Acid Motifs↗

PubMed1998

Cloning and characterization of GETS-1, a goldfish Ets family member that functions as a transcriptional repressor in muscle.

An Ets transcription factor family member, GETS-1, was cloned from a goldfish retina cDNA library. GETS-1 contains a conserved Ets DNA-binding domain at its N-terminus and is most similar to ternary complex factor (TCF) serum-response-factor protein-1a (SAP-1a). GETS-1 is expressed in many tissues, but is enriched in retina and brain. As with the TCFs SAP-1a and ets-related protein (ERP), overexpression of the GETS-1 promoter suppresses nicotinic acetylcholine receptor epsilon-subunit gene expression in cultured muscle cells. A consensus Ets binding site sequence in the promoter of the epsilon-subunit gene is required for GETS-1-mediated repression. GETS-1 repressor activity is abrogated by overexpression of an activated Ras/mitogen-activated protein kinase (MAP kinase) or by mutation of Ser-405, a MAP kinase phosphorylation site in GETS-1. Fusion proteins created between GETS-1 and the Gal4 DNA-binding domain show that, like other TCFs, GETS-1 contains a C-terminal activation domain that is activated by a Ras/MAP kinase signalling cascade. Interestingly, mutation of Ser-405 located within this activation domain abrogated transcriptional activation of the fusion protein.

Amino Acid Sequence↗

PubMed1998

MDM2 expression during mouse embryogenesis and the requirement of p53.

We compared mouse embryonic expression of the MDM2 proto-oncogene, p21WAF1/CIP1 and their transcriptional regulator, p53. MDM2 expression is ubiquitous from 7.5 to 11.5 days post coitum (dpc) and more restricted from 12.5 dpc, with the highest levels in the testes and neural tube. From 14.5 to 18.5 dpc, the nasal respiratory epithelium expresses high levels of MDM2 RNA and protein and p21WAF1/CIP1 RNA, in both wild type and p53 null embryos. MDM2 expression during development is tissue-specific and, like p21WAF1/CIP1, is independent of p53. MDM2 may have a developmental role after 6.5 dpc, when MDM2 null mice die (Jones, S.N., Roe, A.E., Donehower, L.A., Bradley, A., 1995. Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53. Nature 378, 206-208; Montes de Oca Luna, R., Wagner, D.S., Lozano, G., 1995. Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53. Nature 378, 203-206).

Animals↗

PubMed1998

Ets transcription factors: nuclear effectors of the Ras-MAP-kinase signaling pathway.

The Ets family of transcription factors includes nuclear phosphoproteins that are involved in cell proliferation, differentiation and oncogenic transformation. The family is defined by a conserved DNA-binding domain (the ETS-DBD), which forms a highly conserved, winged, helix-turn-helix structural motif. As targets of the Ras-MAPK signaling pathway, Ets proteins function as critical nuclear integrators of ubiquitous signaling cascades. To direct signals to specific target genes, Ets proteins interact with (other) transcription factors that promote the binding of Ets proteins to composite Ras-responsive elements.

Animals↗

PubMed1998

Circulating p53 antibodies as early markers of oral cancer: correlation with p53 alterations.

p53 aberrations are early events in the pathogenesis of betel- and tobacco-related oral malignancies. Accumulation of p53 protein in oral lesions may elicit a humoral immune response against p53 protein in these patients. p53 antibodies (Abs) were analyzed in 183 sera obtained from patients with premalignant or malignant oral lesions and normal individuals by enzyme-linked immunoassay using recombinant p53 protein as antigen. These results were correlated with accumulation of p53 protein in patients' matched oral tissue specimens. Circulating p53 Abs were observed in 24 of 70 (34%) cancer patients and 15 of 50 (30%) patients with premalignant oral lesions. p53 Abs showed a significant association with increase in tumor size and dedifferentiation of tumors, factors indicative of poor prognosis. Expression of p53 protein was analyzed in 43 matched oral lesions (18 premalignant and 25 malignant cases). All the p53-seropositive patients (7 leukoplakia and 11 squamous cell carcinoma) showed elevated levels of p53 protein in matched oral lesions. However, the total number of patients seropositive for p53 Abs was lesser than that of patients exhibiting p53 protein accumulation in oral lesions. Four of the 63 normal healthy individuals who were heavy consumers of tobacco (smoking/chewing) and betel were found to be positive for p53 Abs. Detection of circulating p53 Abs in patients with premalignant oral lesions suggests that humoral immune response against p53 protein is an early event in oral oncogenesis and may be a surrogate marker for both p53 alteration and preclinical cancer.

Adult↗

PubMed1997

The MDM2 C-terminal region binds to TAFII250 and is required for MDM2 regulation of the cyclin A promoter.

MDM2 proto-oncogene expression is aberrant in many human tumors. Its normal role is to modulate the functions of p53. The N terminus of MDM2 interacts with p53, whereas the properties of the rest of the molecule are poorly understood. We show that MDM2 binds to the general transcription factor TFIID in vivo. The C-terminal Ring finger interacts with TAFII250/CCG1, and the central acidic domain interacts with TBP. Expression of MDM2 activates the cyclin A gene promoter but not c-fos, showing that the effects of MDM2 are specific. Deletion of the C-terminal region of MDM2 abolishes activation, showing that the C-terminal domain of MDM2 is functionally important. We found that increasing MDM2 expression to higher levels inhibits the cyclin A promoter. Inhibition appears to result from titration of general transcription factors because MDM2 overexpression inhibits c-fos as well as other promoters in vivo and basal transcription in vitro. The mechanisms of repression of the cyclin A and fos promoters appear to be different. Cyclin A repression is lost by deleting the C terminus, whereas that of c-fos is lost by removal of the acidic domain. These results reinforce the conclusion that the C terminus of MDM2 mediates effects on the cyclin A promoter. MDM2 transformed cells contain elevated levels of cyclin A mRNA, showing that activation occurs under physiological conditions. There is a positive correlation between MDM2 binding to TAFII250 and MDM2 activation of the cyclin A promoter. The C-terminal region of MDM2, which contains the Ring finger, interacts with TAFII250 and is required for regulation of the cyclin A promoter by MDM2. Our results link the activity of MDM2, a transforming protein implicated in many human tumors, with cyclin A, a regulator of the cell cycle.

Amino Acid Sequence↗

PubMed1997

Modulation of ETS-1 transcriptional activity by huUBC9, a ubiquitin-conjugating enzyme.

Ets transcription factors have Ets DNA binding domains, that have a winged helix-turn-helix structure. ETS-1, the founding member of the family, is regulated by the Ras and Ca2+ signaling pathways and is implicated in various physiological processes leading to cell growth, differentiation and apoptosis. We have identified ETS-1 interacting factors with a yeast two-hybrid screen. The majority of the positive clones turned out to encode the human homologue of the yeast ubiquitin-conjugating enzymes UBC9 and Hus5. In two different yeast assays, ETS-1 interacted with huUBC9. In an in vitro GST 'pull-down' assay, ETS-1 and several other Ets family members complexed with huUBC9. Interestingly, in mammalian cells, coexpression of huUBC9 resulted in a substantial increase in the transcriptional activity of ETS-1. Coexpressed huUBC9 did not affect the ETS-1 protein level, and moreover, a point mutation at Cys93, an amino acid known to be essential for ubiquitination, did not abolish the stimulation of the ETS-1 transcriptional activity. Our results indicate that the modulation of ETS-1 activity by huUBC9 results from processes other than ubiquitination and ETS-1 stabilization.

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

Net-b, a Ras-insensitive factor that forms ternary complexes with serum response factor on the serum response element of the fos promoter.

The Ras signalling pathway targets transcription factors such as the ternary complex factors that are recruited by the serum response factor to form complexes on the serum response element (SRE) of the fos promoter. We have identified a new ternary complex factor, Net-b. We report the features of the net gene and show that it produces several splice variants, net-b and net-c. net-b RNA and protein are expressed in a variety of tissues and cell lines. net-c RNA is expressed at low levels, and the protein was not detected, raising the possibility that it is a cryptic splice variant. We have studied the composition of ternary complexes that form on the SRE of the fos promoter with extracts from fibroblasts (NIH 3T3) cultured under various conditions and pre-B cells (70Z/3) before and after differentiation with lipopolysaccharide (LPS). The fibroblast complexes contain mainly Net-b followed by Sap1 and Elk1. Net-b complexes, as well as Sap1 and Elk1, are induced by epidermal growth factor (EGF) stimulation of cells cultured in low serum. Pre-B-cell complexes contain mainly Sap1, with less of Net-b and little of Elk1. There is little change upon LPS-induced differentiation compared to the increase with EGF in fibroblasts. We have also found that Net-b is a nuclear protein that constitutively represses transcription. Net-b is not activated by Ras signalling, in contrast to Net, Sap1a, and Elk1. We have previously reported that down-regulation of Net proteins with antisense RNA increases SRE activity. The increase in SRE activity is observed at low serum levels and is even greater after serum stimulation, showing that the SRE is under negative regulation by Net proteins and the level of repression increases during induction. Net-b, the predominant factor in ternary complexes in fibroblasts, may both keep the activity of the SRE low in the absence of strong inducing conditions and rapidly shut the activity off after stimulation.

3T3 Cells↗

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↗

PubMed1997

Transcription abnormalities potentiate apoptosis of normal human fibroblasts.

BACKGROUND: Apoptosis is a natural process by which damaged and potentially tumorigenic cells are removed. Induction of apoptosis is important in chemotherapy aimed at eliminating cancer cells. We address the mechanisms by which this process can be triggered in cells that are recalcitrant to cell death induced by DNA-damaging agents. MATERIALS AND METHODS: Normal human fibroblasts and lymphoblasts, and fibroblasts with defined genetic changes, were treated with DNA-damaging agents and inhibitors of transcription. Western blotting was used to study the expression of some of the key factors involved in the response to DNA damage and the induction of apoptosis, namely, p53, p21WAFI,Cip1, Mdm2, Bax, and CD95 (Fas/APO1). Apoptosis was followed by various criteria, including DNA fragmentation, specific proteolysis, cell morphology, viability, and FACS scan for sub-G1 cells. RESULTS: Normal human fibroblasts were more resistant than lymphoblasts to DNA damage-induced apoptosis. The DNA-damaging agents mitomycin C and cisplatin induced rapid apoptosis of fibroblasts with defects in the repair of transcribed DNA, compared with wild-type cells or those with defects in overall genome repair. Short-term treatment with inhibitors of RNA polymerase II transcription, actinomycin D, and alpha-amanitin induced rapid cell death of normal fibroblasts. These results show that there is a link between defective transcription and apoptosis. Treatments and genetic backgrounds that favored apoptosis were associated with efficient and prolonged induction of p53 and often altered or imbalanced expression of its downstream effectors p21WAFI,Cip1 and Mdm2, whereas there were no changes in Bax or CD95 (Fas/APO1). CONCLUSION: Transcription inhibitors increase p53 levels and are better inducers of apoptosis than DNA-damaging agents in some cell types. Apoptosis might be triggered by blocked polymerases and/or faulty expression of downstream effectors.

Amanitins↗

PubMed1997

The apoptotic and transcriptional transactivation activities of p53 can be dissociated.

Previous studies have shown that the apoptotic response of cells following DNA damage requires p53 expression. Wild-type p53 protein levels increase in response to DNA damage and its growth-suppressive action is thought to be mediated by transcriptional activation of the p21/WAF1/CIP1 gene, the product of which is a potent inhibitor of cyclin-dependent kinases. The mechanism by which elevated p53 levels lead to apoptosis is not known, but is believed to involve transcriptional activation of apoptotic genes, such as BAX. We have studied transformed human cells that constitutively express high levels of the R273H mutant p53, which has been reported to lack transcriptional activation activity. We used the inability to induce the p21/Waf1/Cip1 protein as a marker to verify the lack of transcriptional activation activity. Cells expressing the R273H mutant of p53 do not show an increase in p21/Waf1/Cip1 following irradiation with ionizing or UVB radiation. Surprisingly, these cells are very susceptible to induction of apoptosis by UVB radiation, as seen by the formation of a nucleosomal ladder and the proteolytic cleavage of poly(ADP-ribose) polymerase. This suggests that the R273 mutant p53 can function normally in apoptosis but not in transcriptional activation following DNA damage. Furthermore, an inhibitor of RNA polymerase II is a potent inducer of apoptosis in these cells, demonstrating that transcription is not required for apoptosis and suggesting that stalled RNA polymerase II complexes can initiate apoptosis. Interestingly, proteolytic cleavage of p53 occurs during apoptosis in these cells, generating a 45-kDa fragment and liberating the DNA repair helicase binding domain of p53. We propose that the peptide liberated from the carboxy terminus of p53 may contribute to its apoptotic activity, possibly through interaction with the XPB and XPD DNA helicases.

Animals↗

PubMed1996

Net (ERP/SAP2) one of the Ras-inducible TCFs, has a novel inhibitory domain with resemblance to the helix-loop-helix motif.

The three ternary complex factors (TCFs), Net (ERP/ SAP-2), ELK-1 and SAP-1, are highly related ets oncogene family members that participate in the response of the cell to Ras and growth signals. Understanding the different roles of these factors will provide insights into how the signals result in coordinate regulation of the cell. We show that Net inhibits transcription under basal conditions, in which SAP-1a is inactive and ELK-1 stimulates. Repression is mediated by the NID, the Net Inhibitory Domain of about 50 amino acids, which autoregulates the Net protein and also inhibits when it is isolated in a heterologous fusion protein. Net is particularly sensitive to Ras activation. Ras activates Net through the C-domain, which is conserved between the three TCFs, and the NID is an efficient inhibitor of Ras activation. The NID, as well as more C-terminal sequences, inhibit DNA binding. Net is more refractory to DNA binding than the other TCFs, possibly due to the presence of multiple inhibitory elements. The NID may adopt a helix-loop-helix (HLH) structure, as evidenced by homology to other HLH motifs, structure predictions, model building and mutagenesis of critical residues. The sequence resemblance with myogenic factors suggested that Net may form complexes with the same partners. Indeed, we found that Net can interact in vivo with the basic HLH factor, E47. We propose that Net is regulated at the level of its latent DNA-binding activity by protein interactions and/or phosphorylation. Net may form complexes with HLH proteins as well as SRF on specific promotor sequences. The identification of the novel inhibitory domain provides a new inroad into exploring the different roles of the ternary complex factors in growth control and transformation.

3T3 Cells↗

PubMed1996

Pax-5 (BSAP) recruits Ets proto-oncogene family proteins to form functional ternary complexes on a B-cell-specific promoter.

The paired box transcription factor Pax-5 (B-cell-specific activator protein) is a key regulator of lineage-specific gene expression and differentiation in B-lymphocytes. We show that Pax-5 functions as a cell type-specific docking protein that facilitates binding of the early B-cell-specific mb-1 promoter by proteins of the Ets proto-oncogene family. Transcriptional activity of the mb-1 promoter in pre-B-cells is critically dependent on binding sites for Pax-5:Ets complexes. Ternary complex assembly requires only the Pax-5 paired box and ETS DNA-binding domains. Mutation of a single base pair in the ternary complex binding site allows for independent binding by Ets proteins but, conversely, inhibits the binding of Pax-5 by itself. Strikingly, the mutation reverses the pattern of complex assembly: Ets proteins recruit Pax-5 to bind the mutated sequence. Recruitment of Net and Elk-1, but not SAP1a, by Pax-5 defines a functional difference between closely related Ets proteins. Replacement of a valine (V68) in the ETS domain of SAP1a by aspartic acid (as found in c-Ets-1, Elk-1, and Net) enhanced ternary complex formation by more than 60-fold. Together, these observations define novel transcription factor interactions that regulate gene expression in B cells.

Amino Acid Sequence↗

PubMed1996

Functional interactions between p53 and the TFIIH complex are affected by tumour-associated mutations.

The p53 tumour suppressor is mutated in the majority of human tumours. p53's proposed role as the guardian of the genome is reflected in its multiple effects on transcription genome stability, cell growth and survival. We show that p53 interacts both physically and functionally with the TFIIH complex. There are multiple protein-protein contacts, involving two regions of p53 and three subunits of TFIIH, ERCC2 (XPD), ERCC3 (XPB) and p62. p53 and its C-terminus (amino acids 320-393) inhibit both of the TFIIH helicases and in vitro transcription in the absence of TFIIH. Transcription inhibition is overcome by TFIIH. The N-terminal region of p53 (1-320), lacking the C-terminus, is inactive on its own, yet apparently affects the activity of the C-terminus in the native protein. Interestingly, mutant p53s that are frequently found in tumours are less efficient inhibitors of the helicases and transcription. We hypothesize that the interactions provide an immediate and direct link for p53 to the multiple functions of TFIIH in transcription, DNA repair and possibly the cell cycle.

Animals↗

PubMed1995

MDM2 transformation in the absence of p53 and abrogation of the p107 G1 cell-cycle arrest.

The p53 tumour-suppressor guards the genome in response to genotoxic stress by transcriptional regulation of genes involved in cell-cycle control, DNA replication, repair and apoptosis such as p21, GADD45, bax and mdm2 (Cox and Lane, 1995). Mdm2 is classically considered to be an inhibitor of p53, that forms an auto-regulatory loop (Momand et al., 1992; Oliner et al., 1993; Wu et al., 1993; Chen et al., 1994; Chen and Levine, 1995). It immortalises cells containing wild type p53 and transforms them together with Ras (Finlay, 1993). We show that, in the absence of p53, mdm2 confers a growth advantage to cells (i.e. "transforms" them) and can overcome a G1 cell-cycl arrest induced by p107, a member of the pRb tumour-suppressor family (Adams and Kaelin, 1995). The minimum "transforming" and p107 inhibiting region of Mdm2 corresponds to its p53 binding domain. p53 inhibits transformation by Mdm2, apparently without requiring transcription. p53 can be considered to be a suppressor of Mdm2, a positive effector of the cell cycle. Mdm2 over-expression in tumours is reminiscent of p53 mutations with gain of function, in that Mdm2 both transforms cells and inhibits p53 activity.

Cell Adhesion↗