In a genome-wide screen for putative tumor suppressor genes, the EBF3 locus on the human chromosome 10q26.3 was found to be deleted or methylated in 73% of the examined cases of brain tumors. EBF3 is expressed in normal brain but is silenced in brain tumors. Therefore, it is suggested that EBF3 is a tumor suppressor. However, it remains unknown whether inactivation of EBF3 locus also occurs in other types of tumors and what functions of EBF3 underlie EBF3-mediated tumor suppression. We show here that expression of EBF3 resulted in cell cycle arrest and apoptosis. The expression of cyclindependent kinase inhibitors was profoundly affected with early activation and then repression of p21 cip1/waf1 and persistent activation of both p27 kip1 and p57 kip2 , whereas genes involved in cell survival and proliferation were suppressed. EBF3 bound directly to p21 cip1/waf1 promoter and regulated transcription from both p21 cip1/waf1 and p27 kip1 promoters in reporter assays. Apoptosis occurred 48 hours after EBF3 expression with caspase-3 activation. Silencing of the EBF3 locus was observed in brain, colorectal, breast, liver, and bone tumor cell lines and its reactivation was achieved on treatment with 5-aza-2 ¶-deoxycytidine and trichostatin A in a significant portion of these tumor cells. Therefore, EBF3 regulates a transcriptional program underlying a putative tumor suppression pathway.
p53 SUMOylation promotes its nuclear export. The SIM-binding groove of a SUMO moiety linked to p53 and a SIM in CRM1 regulates their interaction. CRM1 binds to tetrameric p53 with a properly folded core domain, and CRM1 with a mutated SIM in the HEAT9 loop accumulates with SUMOylated p53 at NPCs and cytoplasmic aggregates.
Daxx is essential for embryonic development and implicated in apoptosis and transcriptional regulation. It is found only in the animal kingdom and appears to arise first in insects. In the Drosophila genus, the Daxx orthologs are much larger than those in other species. Here we show that in addition to a conserved core of ~200 residues, Daxx possesses several conserved domains and two essentially invariable short SUMO-interacting motifs (SIMs), each located at one or the other terminus of the protein.
In an attempt to develop high producing mammalian cell lines expressing glucagon-like-peptide-1-antibody fusion proteins (GLP-1), we have noted that the N-terminal GLP-1 portion of the fusion protein was susceptible to proteolytic degradation during cell culture, which resulted in an inactive product. The majority of the N-terminal clipped product appeared to be due to the removal of the entire biologically active peptide (30 amino acids) from the intact molecule. A number of parameters that influenced the degradative process were investigated. Additionally, protease inhibitors specific for each class of protease were tested. Results suggested that one or more serine-threonine class of protease(s) were involved in this process and inhibitors that are specific for this class of protease, including benzamidine hydrochloride could significantly inhibit the proteolytic degradation of the fusion proteins. Identification of the specific proteases involved in this process by shotgun proteomics methodology will pave the way for engineering the CHOK1SV cell line which will serve as a superior host for the production of future fusion protein products.
The Ad E1B 55-kDa protein (E1B) is a potent transcriptional repressor. In vitro biochemical studies revealed that direct p53-E1B interaction is essential for E1B to block p53-activated transcription and a corepressor may be involved. To understand how E1B represses p53-mediated transcription in vivo, we expressed E1B in several tumor cell lines that express wild type p53. Here we show that E1B strongly suppresses the expression of p53 target genes such as p21 and Puma-␣ in normal growth conditions or after cells were treated with p53-activating chemotherapeutic agents, suggesting that E1B-mediated gene repression is dominant and cannot be reversed via p53 activation. Interestingly, we found that E1B binds to corepressor mSin3A. Mutagenesis analysis indicated that the sequence motif "LHLLA" near the NH 2 terminus of E1B is responsible for mSin3A binding, and this motif is conserved among E1B proteins from different Ad serotypes. The conserved paired amphipathic helix domain 1 of mSin3A is critical for mSin3A-E1B interaction. Surprisingly, E1B mutants that cannot bind to mSin3A can still repress p53 target genes, indicating that it is not the corepressor required for E1B-mediated gene repression. In support of this notion, repression of p53 target genes by E1B is insensitive to HDAC inhibitor trichostatin A. We further show that both the NH 2 -and COOH-terminal domains of E1B are required for the repression function. Therefore, E1B employs a unique repression mechanism to block p53-mediated transcription.The p53 and pRb tumor suppressor pathways are inactivated in virtually all human cancers regardless of their etiology (1, 2). Understanding the precise molecular mechanisms of these pathways is at the center stage of current research efforts in cancer biology. Small DNA tumor viruses such as adenoviruses (Ad), 3 human papillomaviruses, and SV40 can transform cells and cause cancer (3, 4). Each of these viruses produces several proteins that disable both p53 and pRb tumor suppressor pathways in infected cells. Ad E1A proteins physically associate with pRb and release it from E2F transcription factors that activate expression of genes required for DNA replication and cell cycle progression. The Ad 1B region encodes two major proteins in two overlapping ORFs: E1B 55-and 19-kDa, both of which are required for efficient cell transformation. The E1B 19-kDa functions as an inhibitor of apoptosis by binding to proapoptotic proteins Bax and Bak (5), and E1B 55-kDa (hereafter called E1B) participates in transformation by inactivating the p53 pathway (6).Several functions of E1B contribute to the inhibition of p53. Apart from sequestration of p53 in the cytoplasm that blocks p53-mediated apoptosis (7), E1B can inhibit acetylation of p53 and disrupt the interaction between p53 and coactivator p300/ CBP-associated protein (8). Early studies clearly showed that the transcriptional repression function of E1B, but not p53-E1B interaction per se, is critical for Ad-mediated cell transformation (9, 10). These studies demonstrated t...
Adenovirus E1B-55K represses p53-mediated transcription. However, the phenotypic consequence of p53 inhibition by E1B-55K for cell cycle regulation and drug sensitivity in tumor cells has not been examined. In HCT116 cells with constitutive E1B-55K expression, the activation of p53 target genes such as the p21, Mdm2, and Puma genes was attenuated, despite markedly elevated p53 protein levels. HCT116 cells with E1B-55K expression displayed a cell cycle profile similar to that of the isogenic HCT116p53 ؊/؊ cells, including unhindered S-phase entry despite DNA damage. Surprisingly, E1B-55K-expressing cells were more sensitive to drug treatment than parental cells. Compared to HCT116 cells, HCT116p53؊/؊ cells were more susceptible to both doxorubicin and etoposide, and E1B-55K expression had no effects on drug treatment. E1B-55K expression increased the rate of cell proliferation in HCT116 but not in HCT116p53؊/؊ cells. Thus, deregulation of p53-mediated cell cycle control by E1B-55K probably underlies sensitization of HCT116 cells to anticancer drugs. Consistently, E1B-55K expression in A549, A172, and HepG2 cells, all containing wild-type (wt) p53, also enhanced etoposide-induced cytotoxicity, whereas in p53-null H1299 cells, E1B-55K had no effects. We generated several E1B-55K mutants with mutations at positions occupied by the conserved Phe/Trp/His residues. Most of these mutants showed no or reduced binding to p53, although some of them could still stabilize p53, suggesting that binding might not be essential for E1B-55K-induced p53 stabilization. Despite heightened p53 protein levels in cells expressing certain E1B-55K mutants, p53 activity was largely suppressed. Furthermore, most of these E1B-55K mutants could sensitize HCT116 cells to etoposide and doxorubicin. These results indicate that E1B-55K might have utility for enhancing chemotherapy.
<div>Abstract<p>In a genome-wide screen for putative tumor suppressor genes, the EBF3 locus on the human chromosome 10q26.3 was found to be deleted or methylated in 73% of the examined cases of brain tumors. EBF3 is expressed in normal brain but is silenced in brain tumors. Therefore, it is suggested that EBF3 is a tumor suppressor. However, it remains unknown whether inactivation of EBF3 locus also occurs in other types of tumors and what functions of EBF3 underlie EBF3-mediated tumor suppression. We show here that expression of EBF3 resulted in cell cycle arrest and apoptosis. The expression of cyclin-dependent kinase inhibitors was profoundly affected with early activation and then repression of p21<sup>cip1/waf1</sup> and persistent activation of both p27<sup>kip1</sup> and p57<sup>kip2</sup>, whereas genes involved in cell survival and proliferation were suppressed. EBF3 bound directly to p21<sup>cip1/waf1</sup> promoter and regulated transcription from both p21<sup>cip1/waf1</sup> and p27<sup>kip1</sup> promoters in reporter assays. Apoptosis occurred 48 hours after EBF3 expression with caspase-3 activation. Silencing of the EBF3 locus was observed in brain, colorectal, breast, liver, and bone tumor cell lines and its reactivation was achieved on treatment with 5-aza-2′-deoxycytidine and trichostatin A in a significant portion of these tumor cells. Therefore, EBF3 regulates a transcriptional program underlying a putative tumor suppression pathway. (Cancer Res 2006; 66(19): 9445-52)</p></div>
Supplementary Figures 3-6 from An EBF3-Mediated Transcriptional Program That Induces Cell Cycle Arrest and Apoptosis
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