Adenovirus E1A Protein Research Articles

Amplification of inflammation in emphysema and its association with latent adenoviral infection.

This study examines the hypothesis that the cigarette smoke-induced inflammatory process is amplified in severe emphysema and explores the association of this response with latent adenoviral infection. Lung tissue from patients with similar smoking histories and either no (n = 7), mild (n = 7), or severe emphysema (n = 7) was obtained by lung resection. Numbers of polymorphonuclear cells (PMN), macrophages, B cells, CD4, CD8 lymphocytes, and eosinophils present in tissue and airspaces and of epithelial cells expressing adenoviral E1A protein were determined using quantitative techniques. Severe emphysema was associated with an absolute increase in the total number of inflammatory cells in the lung tissue and airspaces. The computed tomography (CT) determined extent of lung destruction was related to the number of cells/m(2) surface area by R(2) values that ranged from 0.858 (CD8 cells) to 0.483 (B cells) in the tissue and 0.630 (CD4 cells) to 0.198 (B cells) in the airspaces. These changes were associated with a 5- to 40-fold increase in the number of alveolar epithelial cells expressing adenoviral E1A protein in mild and severe disease, respectively. We conclude that cigarette smoke-induced lung inflammation is amplified in severe emphysema and that latent expression of the adenoviral E1A protein expressed by alveolar epithelial cells influenced this amplification process.

RACK1 Interacts with E1A and Rescues E1A-induced Yeast Growth Inhibition and Mammalian Cell Apoptosis

The adenoviral E1A proteins are able to promote proliferation and transformation, inhibit differentiation, induce apoptosis, and suppress tumor growth. The extreme N terminus and conserved region one of E1A, which are indispensable for transcriptional regulation and for binding to p300/CBP, TBP, and pCAF, play essential roles in these abilities. These observations strongly suggest an intrinsic link between E1A-mediated transcriptional regulation and other effects. In this report, we show that E1A inhibits the normal growth of Saccharomyces cerevisiae HF7c, and this inhibition also depends on the domains required for transcriptional regulation. We demonstrate that E1A associates with histone acetyltransferase activity and represses the transactivation activity of transcription factor in S. cerevisiae, suggesting that E1A may suppress the expression of genes required for normal growth. Based on yeast growth rescue, we present a genetic screening strategy that identified RACK1 as an E1A antagonizing factor. Expression of human RACK1 efficiently relieves E1A-mediated growth inhibition in HF7c and protects human tumor cells from E1A-induced apoptosis. Finally, we show that RACK1 decreases E1A-associated histone acetyltransferase activity in yeast and mammalian cells, and physically interacts with E1A. Our data demonstrate that RACK1 is a repressor of E1A, possibly by antagonizing the effects of E1A on host gene transcription.

Retinoblastoma-binding Protein 2 (Rbp2) Potentiates Nuclear Hormone Receptor-mediated Transcription

Retinoblastoma-binding protein 2 (Rbp2) was originally identified as a retinoblastoma protein (RB) pocket domain-binding protein. Although Rbp2 has been shown to interact with RB, p107, TATA-binding protein, and T-cell oncogene rhombotin-2, the physiological function of Rbp2 remains unclear. Here we demonstrate that Rbp2 not only binds to nuclear receptors (NRs) but also enhances the transcription mediated by them. Rbp2 interacts with the DNA-binding domains of NRs and potentiates NR-mediated transcription in an AF-2-dependent manner. Both the N-terminal and C-terminal domains of Rbp2 are critical for the transactivation activity of Rbp2 on NRs. The C terminus is the NR-interacting region. In addition, RB functions in maximizing the effect of Rbp2 on the transcription by NRs. These results suggest that Rbp2 is a coregulator of NRs and define a potential role for Rbp2 in NR-mediated transcription.

CREB-binding protein and histone deacetylase regulate the transcriptional activity of Kaposi's sarcoma-associated herpesvirus open reading frame 50.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) open reading frame 50 (ORF50) encodes a viral transcriptional activator, which binds to the KSHV promoter and stimulates the transcription of viral early and late genes, thus activating the lytic cycle of KSHV. We report here that KSHV ORF50 binds to the cellular proteins CREB-binding protein (CBP) and histone deacetylase (HDAC) and these binding events modulate ORF50-activated viral transcription. Binding of ORF50 to CBP and HDAC activates and represses, respectively, ORF50-mediated viral transcription. KSHV ORF50 was shown to bind to the C/H3 domain and the C-terminal transcriptional activation domain of CBP, while CBP bound to the amino-terminal basic domain and the carboxyl-terminal transactivation domain of ORF50. The LXXLL motif within the transcriptional activation domain of ORF50 is reminiscent of the CBP-binding sequence found in nuclear receptor proteins. The adenovirus E1A protein, which also binds to the C/H3 domain of CBP, repressed the transcriptional activation activity of ORF50. The cellular protein c-Jun, which binds to the kinase-induced activation domain of ORF50, stimulated ORF50-mediated viral transcription. The HDAC1-interacting domain of ORF50 was shown to be a central proline-rich sequence. Our data provide a framework for delineating the regulatory mechanisms used by KSHV to modulate its transcription and replication through interaction with both histone acetyltransferases and HDACs.

Plant orthologs of p300/CBP: conservation of a core domain in metazoan p300/CBP acetyltransferase-related proteins.

p300 and CBP participate as transcriptional coregulators in the execution of a wide spectrum of cellular gene expression programs controlling cell differentiation, growth and homeostasis. Both proteins act together with sequence-specific transcription factors to modify chromatin structure of target genes via their intrinsic acetyltransferase activity directed towards core histones and some transcription factors. So far, p300-related proteins have been described in animals ranging from Drosophila and Caenorhabditis elegans to humans. In this report, we describe p300/CBP-like polypeptides in the plant Arabidopsis thaliana. Interestingly, homology between animal and plant p300/CBP is largely restricted to a C-terminal segment, about 600 amino acids in length, which encompasses acetyltransferase and E1A-binding domains. We have examined whether this conservation in sequence is paralleled by a conservation in function. The same amino acid residues critical for acetyltransferase activity in human p300 are also critical for the function of one of the plant orthologs. Remarkably, plant proteins bind to the adenovirus E1A protein in a manner recapitulating the binding specificity of mammalian p300/CBP. The striking conservation of an extended segment of p300/CBP suggests that it may constitute a functional entity fulfilling functions that may be essential for all metazoan organisms.

DNA replication blockade impairs p53-transactivation.

Tumor-suppressor genes are necessary for maintaining cell growth control. When these genes fail to function properly, deregulated growth—a defining feature of cancer cells—ensues. The p53 tumor suppressor, first described in 1979 as an interacting protein with the transforming large T-antigen of simian virus 40, has emerged as the most commonly altered gene and growth suppressing pathway in human cancer. p53 protein is stabilized in response to a number of stresses, including exposure of cells to DNA damaging agents, hypoxia, or inappropriate expression or activation of a number of cellular or viral oncogenes. The upstream regulatory interactions and downstream events that follow from p53 stabilization provide a complex network of signals ultimately leading to cell cycle arrest, apoptosis, and tumor suppression (1, 2). We know relatively little, however, about the cell cycle dependence of the p53 response, and the role of p53 in the response to replication blockade in S-phase of the cell cycle. Studies reported by Gottifredi et al. (3) in this issue of PNAS shed light on the p53 response in S-phase, differentiate between passage through S-phase and blockade in S-phase in terms of the events downstream of p53 activation, and provide evidence that the transcriptional program downstream of “activated” p53 is drastically attenuated if DNA synthesis is blocked. Interesting questions arise regarding how and why cells stabilize and inhibit p53 during S-phase blockade and parallels can be drawn to the transforming adenovirus E1A protein or the hypoxia inducible factor activating drug desferoxamine, both of which also stabilize p53 while inhibiting its transactivation potential.

Tumor suppression activity of adenovirus e1a protein: Anoikis and the epithelial phenotype

Adenovirus E1a proteins reverse-transform diverse human tumor cells in culture. This has stimulated interest in the arenas of clinical and basic cancer research. Clinically, cancer gene therapy trials on E1a are in progress, and drug discovery strategies based on E1a are being considered. Biologically, the effect of E1a is unique in that it overrides most or all oncogenic signaling pathways to yield nontumorigenic cells. Apparently, this is a consequence of the ability of E1a to reprogram transcription in tumor cells so as to produce an epithelial phenotype that is refractory to oncogenic growth stimulation. The molecular basis for this effect is emerging.

The Interaction of the Carboxyl Terminus-binding Protein with the Smad Corepressor TGIF Is Disrupted by a Holoprosencephaly Mutation in TGIF

The homeodomain protein TGIF represses transcription in part by recruiting histone deacetylases. TGIF binds directly to DNA to repress transcription or interacts with TGF-beta-activated Smads, thereby repressing genes normally activated by TGF-beta. Loss of function mutations in TGIF result in holoprosencephaly (HPE) in humans. One HPE mutation in TGIF results in a single amino acid substitution in a conserved PLDLS motif within the amino-terminal repression domain. We demonstrate that TGIF interacts with the corepressor carboxyl terminus-binding protein (CtBP) via this motif. CtBP, which was first identified by its ability to bind the adenovirus E1A protein, interacts both with gene-specific transcriptional repressors and with a subset of polycomb proteins. Efficient repression of TGF-beta-activated gene responses by TGIF is dependent on interaction with CtBP, and we show that TGIF is able to recruit CtBP to a TGF-beta-activated Smad complex. Disruption of the PLDLS motif in TGIF abolishes the interaction of CtBP with TGIF and compromises the ability of TGIF to repress transcription. Thus, at least one HPE mutation in TGIF appears to prevent CtBP-dependent transcriptional repression by TGIF, suggesting an important developmental role for the recruitment of CtBP by TGIF.

Cyclin D1 suppresses retinoblastoma protein-mediated inhibition of TAFII250 kinase activity.

The retinoblastoma tumor suppressor protein has been shown to bind directly and inhibit a transcriptionally-important amino-terminal kinase domain of TATA-binding protein-associated factor TAFII250. Cyclin D1 also is able to associate with the amino terminus of TAFII250 in a region very similar to or overlapping the Rb-binding site. In this study, we have examined whether cyclin D1 affects the functional interaction between Rb and TAFII250. We observed that when cyclin D1 is coincubated with Rb and TAFII250, the ability of Rb to inhibit TAFII250 kinase activity is effectively blocked. However, cyclin D1 by itself has no apparent effect on TAFII250 kinase activity. We further found that the Rb-related protein p107 can inhibit TAFII250 kinase activity, and this inhibition is likewise alleviated by cyclin D1. Cyclin D1 prevents the kinase-inhibitory effect of an Rb mutant unable to bind to D-type cyclins, indicating that it is acting through its association with TAFII250 and not with Rb. However, we found no evidence of TAFII250-binding competition between Rb and cyclin D1 in vitro. The adenovirus E1A protein, which also binds to both Rb and TAFII250, exhibited a suppressive effect on Rb-mediated kinase inhibition similar to that seen with cyclin D1. Our results suggest a novel means by which cyclin D1 may be able to independently regulate the activity of Rb.

Regulation of Lef-mediated Transcription and p53-dependent Pathway by Associating β-Catenin with CBP/p300

CBP and its homologue p300 play significant roles in cell differentiation, cell cycle, and anti-oncogenesis. We demonstrated that beta-catenin, recently known as a potent oncogene, and CBP/p300 are associated through its CH3 region, which is a primary target of adenoviral oncoprotein E1A and various nuclear proteins, such as p53, cyclin E, and AP-1, and both are colocalized in the nuclear bodies. CBP/p300 potentiated Lef-mediated transactivation of beta-catenin, and E1A, a potent inhibitor of CBP/p300, repressed its transactivation. Furthermore, overexpression of stable beta-catenin mutant competitively suppressed the p53-dependent pathway. These may be a key mechanism of beta-catenin involved in oncogenic events underlying disruption of tumor suppressor function through CBP/p300.

Role for p300 in Pax 8 Induction of Thyroperoxidase Gene Expression

The nuclear p300 protein functions as a co-activator of gene transcription. Here we show that p300 works as a co-activator of the transcription factor Pax 8 on the thyroperoxidase gene promoter. Consistent with its role as co-activator, p300 potentiates Pax 8-activated transcription. Furthermore, we provide evidence supporting the formation of a complex between both factors in vivo and in vitro. This interaction involves the amino-terminal and CH3 domains of p300 and the trans-activation domain of Pax 8 at its carboxyl-terminal end. We show that the CH3 domain is crucial for the co-activator role of p300 on the thyroperoxidase gene promoter. In agreement with our finding and with the ability of the adenoviral protein E1A to bind p300, we show that E1A down-regulates Pax 8 activity.

Dissimilar Immunogenicities of Human Papillomavirus E7 and Adenovirus E1A Proteins Influence Primary Tumor Development

Although human papillomaviruses (HPV) and adenoviruses (Ad) both transform cells by expressing functionally related oncogenes (Ad-E1A/E1B; HPV-E7/E6), only HPV are oncogenic in humans. Prior studies have shown that HPV-transformed cells are resistant to NK cell lysis and E7- and E6-specific CTL are inefficiently generated in women with HPV-induced cervical cancer. Therefore, we postulated that the dissimilar oncogenicities of Ad and HPV may be caused by a protective NK and T cell response that is triggered by transformed cells expressing E1A, but not by E7. To test this hypothesis, mice that were either immunologically intact, lacked T cells, or lacked both NK and T cells were challenged with Ad serotype 5 (Ad5)-E1A- or HPV16-E7-transfected tumor cells. E7-expressing tumor cells were resistant to NK cell lysis in vitro and failed to elicit a measurable anti-tumor NK or T cell response in vivo. The concomitant expression of E6 did not change this phenotype. In contrast, E1A-expressing tumor cells were sensitive to NK lysis in vitro and triggered a protective NK and T cell immune response in vivo. These data suggest differences in the capacities of E1A or E7 oncoproteins to trigger protective anti-tumor immune responses may contribute to the dissimilar oncogenicities of Ad and HPV in humans.

Transcription Factor ZBP-89 Cooperates with Histone Acetyltransferase p300 during Butyrate Activation of p21 waf1 Transcription in Human Cells

Inducible p53-independent regulation of the cyclin-dependent kinase inhibitor p21(waf1) transcription is mediated through proximal GC-rich sites. Prior studies have shown that Sp1, Sp3, and the histone acetylase co-activator p300 are components of the complexes binding to these sites. Although Sp1 and Sp3 collaborate with p300, a direct interaction between Sp1 and p300 does not occur. This study sought to determine whether ZBP-89 rather than Sp1 is the direct target of p300 during butyrate induction of p21(waf1). ZBP-89 (BFCOL1, BERF-1, ZNF 148) is a Krüppel-type zinc finger transcription factor that binds to GC-rich elements and represses or activates known target genes. Adenoviral-mediated expression of ZBP-89 in HT-29 cells revealed that ZBP-89 potentiates butyrate induction of endogenous p21(waf1) gene expression. Further, cotransfection of a ZBP-89 expression vector with a 2.3-kilobase p21(waf1) reporter recapitulated the potentiation by butyrate. DNase I footprinting analysis of the human p21(waf1) promoter with recombinant ZBP-89 identified a binding site at -245 to -215. Electrophoretic mobility shift assays confirmed that both recombinant and endogenous ZBP-89 and Sp1 bind to this element. The potentiation was abolished in the presence of adenoviral protein E1A. Deletion of the N-terminal domain of ZBP-89 abolished the potentiation mediated by butyrate treatment. This same deletion mutant abolished the ZBP-89 interaction with p300. Cotransfection of p300 with ZBP-89 stimulated the p21(waf1) promoter in the absence of butyrate. p300 co-precipitated with ZBP-89 but not with Sp1, whereas ZBP-89 co-precipitated with Sp1. Together, these findings demonstrate that ZBP-89 also plays a critical role in butyrate activation of the p21(waf1) promoter and reveals preferential cooperation of this four-zinc finger transcription factor with p300.

A CCAAT/Enhancer-binding Protein Site within Antioxidant/Electrophile Response Element Along with CREB-binding Protein Participate in the Negative Regulation of RatGST-Ya Gene in Vascular Smooth Muscle Cells

A CCAAT/Enhancer-binding Protein Site within Antioxidant/Electrophile Response Element Along with CREB-binding Protein Participate in the Negative Regulation of RatGST-Ya Gene in Vascular Smooth Muscle Cells

Inhibition of S-phase progression by adeno-associated virus Rep78 protein is mediated by hypophosphorylated pRb.

Adeno-associated virus (AAV) has an antiproliferative action on cells. We investigated the effect of the AAV replication proteins (Rep) on the cell division cycle using retroviral vectors. Rep78 and Rep68 inhibited the growth of primary, immortalized and transformed cells, while Rep52 and Rep40 did not. Rep68 induced cell cycle arrest in phases G(1) and G(2), with elevated CDK inhibitor p21 and reduced cyclin E-, A- and B1-associated kinase activity. Rep78-expressing cells were also impaired in S-phase progression and accumu lated almost exclusively with hypophosphorylated retinoblastoma protein (pRb). The differences between Rep78 and Rep68 were mapped to the C-terminal zinc finger domain of Rep78. Rep78-induced S-phase arrest could be bypassed by adenoviral E1A or papillomaviral E7 proteins but not by E1A or E7 mutants unable to bind pRb. Rb(-/-) primary mouse embryonic fibroblasts displayed a strongly reduced S-phase arrest when challenged with Rep78, compared with matched Rb(+/+) controls. These results suggest that physiological levels of active pRb can interfere with S-phase progression. We propose that the AAV Rep78 protein arrests cells within S-phase by a novel mechanism involving the ectopic accumulation of active pRb.

C/EBPalpha inhibits cell growth via direct repression of E2F-DP-mediated transcription.

Using an inducible transcription system which allows the regulated expression of C/EBP isoforms in tissue culture cells, we have found that the ectopic expression of C/EBPalpha, at a level comparable to that found in normal liver tissue, has a pronounced antimitogenic effect in mouse L cells and NIH 3T3 cells. The inhibition of cell division by C/EBPalpha in mouse cells cannot be reversed by simian virus 40 T antigen, by oncogenic ras, or by adenovirus E1a protein. When expressed in thymidine kinase-deficient L cells or 3T3 cells, C/EBPalpha is detected in a protein complex which binds to the E2F binding sites found in the promoters of the genes for E2F-1 and dihydrofolate reductase (DHFR). Bacterially expressed C/EBPalpha has no affinity for these E2F sites, but when recombinant C/EBPalpha is added to nuclear extracts from mouse fibroblasts, a new E2F binding activity appears, which contains the C/EBPalpha protein. Using an E2F-DP1-responsive promoter linked to a reporter gene, it can be shown that C/EBPalpha directly inhibits the induction of this promoter by E2F-DP1 in transient-transfection assays. Furthermore, C/EBPalpha can be shown to inhibit the S-phase induction of the E2F and DHFR promoters in permanent cell lines. These findings delineate a straightforward mechanism for C/EBPalpha-mediated cell growth arrest through repression of E2F-DP-mediated S-phase transcription.

Evidence for a function of CtBP in epithelial gene regulation and anoikis.

Previously, we reported that adenovirus E1a protein behaves as a tumor suppressor in human cells. It apparently functions by transcriptionally inducing an array of epithelial cell adhesion genes, while repressing other cell-type specific genes, thus producing an epithelial phenotype. Concomitantly, the cells become sensitive to anoikis (apoptosis of epithelial cells detached from extracellular matrix), potentially causing tumor suppression. E1a protein interacts with the nuclear acetylases p300, CBP and P/CAF, and also with the co-repressor protein CtBP. In this study, we have determined the role of these interactions in E1a's phenotypic effects on human tumor cells. The results indicate that E1a's interaction with CtBP activates at least three epithelial cell adhesion gene promoters. The E-cadherin repressor appeared to be the CtBP-interacting protein delta EF1/ZEB, which bound the ras-repressible E-boxes of the E-cadherin promoter. The E1a-CtBP interaction also contributed to anoikis-sensitization. E1a's interactions with the nuclear acetylases conferred epithelial morphologies but did not activate epithelial genes. These latter interactions did not sensitize tumor cells to anoikis but nevertheless conferred tumor suppression. These results implicate CtBP as an antagonist of the epithelial phenotype and anoikis. They also indicate a new but undefined role for nuclear acetylases in maintaining the transformed phenotype.

Significance of Nuclear Relocalization of ERK1/2 in Reactivation of c-fos Transcription and DNA Synthesis in Senescent Fibroblasts

Two of mitogen-activated protein kinases (MAPK), p44(mapk)/p42(mapk) extracellular signal-regulated kinases (ERK1/2), translocate into nuclei following activation and play critical roles in connecting the signal to gene expression and allowing cell-cycle entry. Here we found that the nuclear translocation of ERK1/2 in response to growth stimuli was significantly inhibited in senescent cells that were irreversibly growth arrested, compared with presenescent cells. The activation step of these enzymes was not impaired, since ERK1/2 were phosphorylated and activated in senescent cells as efficiently as in presenescent cells. By elaborately localizing ERK2 in the nuclei of senescent cells, we could restore c-fos transcriptional activity upon growth stimuli, which was repressed in senescent cells. Furthermore, the nuclear localization of ERK1/2 has been suggested to potentiate the proliferative activity of the senescent cells in collaboration with adenovirus E1A protein. More importantly, SV40 large T antigen, the strong inducer of DNA synthesis, had the inherent ability to restore nuclear relocalization of active ERK1/2 in senescent cells, which was essentially required for the reinitiation of DNA synthesis. Thus, manipulating the relocalization of ERK1/2 into nuclei was expected to open the way to overcome some of the senescent phenotypes.

P21 and retinoblastoma protein control the absence of DNA replication in terminally differentiated muscle cells.

During differentiation, skeletal muscle cells withdraw from the cell cycle and fuse into multinucleated myotubes. Unlike quiescent cells, however, these cells cannot be induced to reenter S phase by means of growth factor stimulation. The studies reported here document that both the retinoblastoma protein (Rb) and the cyclin-dependent kinase (cdk) inhibitor p21 contribute to this unresponsiveness. We show that the inactivation of Rb and p21 through the binding of the adenovirus E1A protein leads to the induction of DNA replication in differentiated muscle cells. Moreover, inactivation of p21 by E1A results in the restoration of cyclin E-cdk2 activity, a kinase made nonfunctional by the binding of p21 and whose protein levels in differentiated muscle cells is relatively low in amount. We also show that restoration of kinase activity leads to the phosphorylation of Rb but that this in itself is not sufficient for allowing differentiated muscle cells to reenter the cell cycle. All the results obtained are consistent with the fact that Rb is functioning downstream of p21 and that the activities of these two proteins may be linked in sustaining the postmitotic state.

Adenoviral protein E1A prevents potentiatiation of butyrate-activated p21 WAFL by ZBP-89 through direct binding

Adenoviral protein E1A prevents potentiatiation of butyrate-activated p21 WAFL by ZBP-89 through direct binding