Cell Transcriptional Machinery Research Articles

Reconstructing differentially co-expressed gene modules and regulatory networks of soybean cells

BackgroundCurrent experimental evidence indicates that functionally related genes show coordinated expression in order to perform their cellular functions. In this way, the cell transcriptional machinery can respond optimally to internal or external stimuli. This provides a research opportunity to identify and study co-expressed gene modules whose transcription is controlled by shared gene regulatory networks.ResultsWe developed and integrated a set of computational methods of differential gene expression analysis, gene clustering, gene network inference, gene function prediction, and DNA motif identification to automatically identify differentially co-expressed gene modules, reconstruct their regulatory networks, and validate their correctness. We tested the methods using microarray data derived from soybean cells grown under various stress conditions. Our methods were able to identify 42 coherent gene modules within which average gene expression correlation coefficients are greater than 0.8 and reconstruct their putative regulatory networks. A total of 32 modules and their regulatory networks were further validated by the coherence of predicted gene functions and the consistency of putative transcription factor binding motifs. Approximately half of the 32 modules were partially supported by the literature, which demonstrates that the bioinformatic methods used can help elucidate the molecular responses of soybean cells upon various environmental stresses.ConclusionsThe bioinformatics methods and genome-wide data sources for gene expression, clustering, regulation, and function analysis were integrated seamlessly into one modular protocol to systematically analyze and infer modules and networks from only differential expression genes in soybean cells grown under stress conditions. Our approach appears to effectively reduce the complexity of the problem, and is sufficiently robust and accurate to generate a rather complete and detailed view of putative soybean gene transcription logic potentially underlying the responses to the various environmental challenges. The same automated method can also be applied to reconstruct differentially co-expressed gene modules and their regulatory networks from gene expression data of any other transcriptome.

Genome-wide expression profiling shows transcriptional reprogramming in Fusarium graminearum by Fusarium graminearum virus 1-DK21 infection

BackgroundFusarium graminearum virus 1 strain-DK21 (FgV1-DK21) is a mycovirus that confers hypovirulence to F. graminearum, which is the primary phytopathogenic fungus that causes Fusarium head blight (FHB) disease in many cereals. Understanding the interaction between mycoviruses and plant pathogenic fungi is necessary for preventing damage caused by F. graminearum. Therefore, we investigated important cellular regulatory processes in a host containing FgV1-DK21 as compared to an uninfected parent using a transcriptional approach.ResultsUsing a 3′-tiling microarray covering all known F. graminearum genes, we carried out genome-wide expression analyses of F. graminearum at two different time points. At the early point of growth of an infected strain as compared to an uninfected strain, genes associated with protein synthesis, including ribosome assembly, nucleolus, and ribosomal RNA processing, were significantly up-regulated. In addition, genes required for transcription and signal transduction, including fungal-specific transcription factors and cAMP signaling, respectively, were actively up-regulated. In contrast, genes involved in various metabolic pathways, particularly in producing carboxylic acids, aromatic amino acids, nitrogen compounds, and polyamines, showed dramatic down-regulation at the early time point. Moreover, genes associated with transport systems localizing to transmembranes were down-regulated at both time points.ConclusionThis is the first report of global change in the prominent cellular pathways in the Fusarium host containing FgV1-DK21. The significant increase in transcripts for transcription and translation machinery in fungal host cells seems to be related to virus replication. In addition, significant down-regulation of genes required for metabolism and transporting systems in a fungal host containing the virus appears to be related to the host defense mechanism and fungal virulence. Taken together, our data aid in the understanding of how FgV1-DK21 regulates the transcriptional reprogramming of F. graminearum.

Role of non‐specific DNA in reducing coding DNA requirement for transient gene expression with CHO and HEK‐293E cells

Transient gene expression (TGE) is a rapid method for the production of recombinant proteins in mammalian cells. While the TGE volumetric productivity has improved significantly over the past decade, the amount of plasmid DNA (pDNA) needed for transfection remains very high. Here, we examined the use of non-specific (filler) DNA to partially replace the transgene-bearing plasmid DNA (coding pDNA) in transfections of Chinese hamster ovary (CHO) and human embryo kidney (HEK-293E) cells. When the optimal amount of coding pDNA for either host was reduced by 67% and replaced with filler DNA, the recombinant protein yield decreased by only 25% relative to the yield in control transfections. Filler DNA did not affect the cellular uptake or intracellular stability of coding pDNA, but its presence lead to increases of the percentage of transfected cells and the steady-state level of transgene mRNA compared to control transfections. Studies of the physicochemical properties of DNA-polyethyleneimine (PEI) complexes with or without filler DNA did not reveal any differences in their size or surface charge. The results suggest that filler DNA allows the coding pDNA to be distributed over a greater number of DNA-PEI complexes, leading to a higher percentage of transfected cells. The co-assembly of filler DNA and coding pDNA within complexes may also allow the latter to be more efficiently utilized by the cell's transcription machinery, resulting in a higher level of transgene mRNA.

Resveratrol, through NF‐Y/p53/Sin3/HDAC1 complex phosphorylation, inhibits estrogen receptor α gene expressionviap38MAPK/CK2 signaling in human breast cancer cells

Agents to counteract acquired resistance to hormonal therapy for breast cancer would substantially enhance the long-term benefits of hormonal therapy. In the present study, we demonstrate how resveratrol (Res) inhibits human breast cancer cell proliferation, including MCF-7 tamoxifen-resistant cells (IC(50) values for viability were in the 30-45 μM range). We show that Res, through p38(MAPK) phosphorylation, causes induction of p53, which recruits at the estrogen receptor α (ERα) proximal promoter, leading to an inhibition of ERα expression in terms of mRNA and protein content. These events appear specifically p53 dependent, since they are drastically abrogated with p53-targeting siRNA. Coimmunoprecipitation assay showed specific interaction between p53, the Sin3A corepressor, and histone deacetylase 1 (HDAC1), which was phosphorylated. The enhancement of the tripartite complex p53/Sin3A/HDAC1, together with NF-Y on Res treatment, was confirmed by chromatin immunoprecipitation analyses, with a concomitant release of Sp1 and RNA polymerase II, thereby inhibiting the cell transcriptional machinery. The persistence of such effects in MCF-7 tamoxifen-resistant cells at a higher extent than parental MCF-7 cells addresses how Res may be considered a useful pharmacological tool to be exploited in the adjuvant settings for treatment of breast cancer developing hormonal resistance.

Sequential changes at differentiation gene promoters as they become active in a stem cell lineage

Transcriptional silencing of terminal differentiation genes by the Polycomb group (PcG) machinery is emerging as a key feature of precursor cells in stem cell lineages. How, then, is this epigenetic silencing reversed for proper cellular differentiation? Here, we investigate how the developmental program reverses local PcG action to allow expression of terminal differentiation genes in the Drosophila male germline stem cell (GSC) lineage. We find that the silenced state, set up in precursor cells, is relieved through developmentally regulated sequential events at promoters once cells commit to spermatocyte differentiation. The programmed events include global downregulation of Polycomb repressive complex 2 (PRC2) components, recruitment of hypophosphorylated RNA polymerase II (Pol II) to promoters, as well as the expression and action of testis-specific homologs of TATA-binding protein-associated factors (tTAFs). In addition, action of the testis-specific meiotic arrest complex (tMAC), a tissue-specific version of the MIP/dREAM complex, is required both for recruitment of tTAFs to target differentiation genes and for proper cell type-specific localization of PRC1 components and tTAFs within the spermatocyte nucleolus. Together, the action of the tMAC and tTAF cell type-specific chromatin and transcription machinery leads to loss of Polycomb and release of stalled Pol II from the terminal differentiation gene promoters, allowing robust transcription.

Research Spotlight: Delivery of Therapeutic RNA Molecules to Cancer Cells By Bacteria

Delivery of RNA-based therapeutics, for example RNA interference (RNAi) effectors, to target cells is one of the major obstacles for the development of RNA-based therapies. Since it has been known for a long time that bacteria can mediate tumor shrinkage, it was obvious to use nonpathogenic bacteria to produce and deliver therapeutic RNA molecules into target cells to induce RNAi. During the last years, two bacteria-based concepts were developed for this strategy, transkingdom RNAi (tkRNAi) and bacteria-mediated RNAi (bmRNAi). The first concept, tkRNAi, delivers RNAi effectors into target cells by invasive bacteria, which themselves produce therapeutic shRNAs. The bmRNAi technology utilizes invasive bacteria conveying RNAi effector-encoding DNA constructs that will act as a matrix for transcription of these sequences in the target cell by the host cell's transcription machinery.

First evidences that Resveratrol through p53/Sin3/HDAC1 complex phosphorylation inhibits ESR1 gene expression via p38MAPK signalling.

Agents to counteract acquired resistance to hormonal therapy for breast cancer would substantially enhance the long-term benefits of hormonal therapy. In the present study we demonstrate how resveratrol (Res) inhibits human breast cancer cell proliferation including MCF-7 tamoxifen-resistant cells. We show that Res, through p38MAPK phosphorylation, causes induction of p53 which recruits at the ER alpha proximal promoter, leading to an inhibition of ER alpha expression in terms of mRNA and protein content. These events appear specifically p53-dependent, since it is drastically abrogated with p53 targeting siRNA. Co-immunoprecipitation of nuclear extracts showed specific interaction between p53, the Sin3A corepressor and HDAC1 which was phosphorylated. In the presence of a specific p38MAPK inhibitor the above described interactions were prevented. The enhancement of the tripartite complex p53/Sin3A/HDAC1 with Res treatment was confirmed by ChIP analyses, with a concomitant release of Sp1 and RNA polymerase II, thereby inhibiting the cell transcriptional machinery. The persistence of such effects in MCF-7 tamoxifen resistant cells at a higher extent than parental MCF-7 cells, addresses how Res may be considered as useful pharmacological tool to be exploited in the adjuvant settings for treatment of breast cancer developing hormonal resistance.

Nuclear Receptor (PXR), Ligand and Co-Activator Interactions Measured by Total Internal Reflection Fluorescence Microscopy

The pregnane X receptor (PXR) is a member of the nuclear receptor family. PXR acts in a ligand-dependent manner, in concert with its dimerization partner, the retinoid X receptor, and co-regulators, to modulate the expression of proteins that metabolize endogenous and exogenous compounds. Here, we report the use of total internal reflection fluorescence microscopy (TIRFM) for examining interactions between the ligand-binding domain of PXR (PXR-LBD), its ligands, and a peptide derived from a co-activator. TIRFM is a surface-specific technique that enables us to study the behavior of fluorescent species close to or at interfaces. Our experimental system consists of biotinylated PXR-LBD immobilized on a fused silica substrate coated with NeutrAvidin and ovalbumin, a ligand (e.g., the antibiotic rifampicin) and a fluorescently labeled, 25 amino acid fragment of the steroid receptor co-activator-1 (F-SRC-1). Using TIRFM, we measured the surface-associated fluorescence as a function of the F-SRC-1 concentration at fixed ligand concentrations. These curves were fit to a model of single-site binding to obtain apparent equilibrium constants. The apparent equilibrium constants as a function of ligand concentration were fit to an appropriate model to obtain binding constants for the ligand/PXR-LBD interaction, F-SRC-1 binding to apo and ligand-bound PXR-LBD and ligand binding to F-SRC-1-bound PXR-LBD. The approach yielded four, previously unmeasured, binding constants. These values indicate that the increase in PXR's affinity for its co-activator peptide upon binding an activating ligand is modest. This observation implies that for gene expression to be significantly upregulated, the signal arising from the activation of PXR must be amplified downstream - possibly when co-activators like SRC-1 start recruiting the cell's transcription machinery. Kinetic data obtained by combining TIR illumination with fluorescence recovery after photobleaching and/or fluorescence correlation spectroscopy may also be discussed.

Ubiquitin-dependent and Ubiquitin-independent Control of Subunit Stoichiometry in the SWI/SNF Complex

The mammalian SWI/SNF chromatin remodeling complex is a key player in multiple chromatin transactions. Core subunits of this complex, including the ATPase, Brg-1, and various Brg-1-associated factors (BAFs), work in concert to maintain a functional remodeling complex. This intra-complex regulation is supervised by protein-protein interactions, as stoichiometric levels of BAF proteins are maintained by proteasomal degradation. We show that the mechanism of BAF155-mediated stabilization of BAF57 involves blocking its ubiquitination by preventing interaction with TRIP12, an E3 ubiquitin ligase. Consequently, as opposed to complexed BAF57, whose principal lysines are unavailable for ubiquitination, uncomplexed BAF57 can be freely ubiquitinated and degraded by the proteasome. Additionally, a BAF57 mutant, which contains no lysine residues, was found to retain its ability to be stabilized by interaction with BAF155, suggesting that in addition to the ubiquitin-dependent mechanism of BAF57 degradation, there exists a ubiquitin-independent mechanism that may involve the direct interaction of BAF57 with the proteasome. We propose that this regulatory mechanism exists to ensure functional fidelity of the complex and prevent the accumulation of uncomplexed proteins, which may disrupt the normal activity of the complex.

Genomic imprinting and cancer: Remembering the parental origin

Genomic imprinting results in only one copy of a diploid pair of alleles being expressed in a parentof-origin-specific manner. The ‘imprint’ encodes a memory of whether a gene came through the maternal or paternal line and contains the information that decides which parental copy will be active or silent. Imprints are established in the developing gametes, passed on to the next generation after fertilization where they are read and maintained in the somatic cells or erased and reset in the germ cells. The components of the ‘memory’ are a combination of epigenetic features such as DNA methylation, post-translational histone modifications and protein/RNA factors that can bind to DNA and label the genes such that a cell's transcription machinery can distinguish between maternal and paternal alleles. Most imprinted genes are associated with sequences that are methylated on only one parental allele, known as differentially methylated regions (DMRs).

Transcriptome analysis of rainbow trout infected with high and low virulence strains of Infectious hematopoietic necrosis virus

There are three main genetic lineages or genogroups of Infectious hematopoietic necrosis virus (IHNV) in N. America. Strains representing the M genogroup are more virulent in rainbow trout relative to the U genogroup. In this study, we used microarray analysis to evaluate potential mechanisms responsible for host-specific virulence in rainbow trout that were given intraperitoneal injections of buffer or a representative M or U type virus strain. Reverse transcriptase quantitative PCR (RT-qPCR) was used to assess viral load and gene expression of select immune genes. Viral load was significantly higher in trout infected with the M virus starting at 24 h post-infection (p.i.) and continuing until 72 h p.i. Microarray analysis of the 48 h time point revealed 153 up-regulated and 248 down-regulated features in response to M virus infection but only 62 up-regulated and 49 down-regulated features following U virus infection. Translation and transcription features were among the most frequent down-regulated features in response to M virus infection and may be associated with the host cell shutoff phenomenon. A greater host cell shutoff response by the M virus may facilitate subversion of the host cell transcriptional machinery and enhance viral replication, suggesting the M virus may be better optimized to manipulate the rainbow trout transcriptional and translational machinery. Anti-viral associated features were the most commonly up-regulated features. A common set of features were up-regulated in both the M and U infection groups, but were induced to a higher magnitude in the M infection group. Gene expression of the anti-viral genes Mx-1 and Vig-1 was correlated but not entirely dependent on viral load in the anterior kidney. Slower replication of the U virus may allow the host more time to induce protective anti-viral immune mechanisms.

Defining the RNA polymerase III transcriptome: Genome-wide localization of the RNA polymerase III transcription machinery in human cells

Our view of the RNA polymerase III (Pol III) transcription machinery in mammalian cells arises mostly from studies of the RN5S (5S) gene, the Ad2 VAI gene, and the RNU6 (U6) gene, as paradigms for genes with type 1, 2, and 3 promoters. Recruitment of Pol III onto these genes requires prior binding of well-characterized transcription factors. Technical limitations in dealing with repeated genomic units, typically found at mammalian Pol III genes, have so far hampered genome-wide studies of the Pol III transcription machinery and transcriptome. We have localized, genome-wide, Pol III and some of its transcription factors. Our results reveal broad usage of the known Pol III transcription machinery and define a minimal Pol III transcriptome in dividing IMR90hTert fibroblasts. This transcriptome consists of some 500 actively transcribed genes including a few dozen candidate novel genes, of which we confirmed nine as Pol III transcription units by additional methods. It does not contain any of the microRNA genes previously described as transcribed by Pol III, but reveals two other microRNA genes, MIR886 (hsa-mir-886) and MIR1975 (RNY5, hY5, hsa-mir-1975), which are genuine Pol III transcription units.

Attenuated Strains of Influenza A Viruses Do Not Induce Degradation of RNA Polymerase II

We have previously shown that infection with laboratory-passaged strains of influenza virus causes both specific degradation of the largest subunit of the RNA polymerase II complex (RNAP II) and inhibition of host cell transcription. When infection with natural human and avian isolates belonging to different antigenic subtypes was examined, we observed that all of these viruses efficiently induce the proteolytic process. To evaluate whether this process is a general feature of nonattenuated viruses, we studied the behavior of the influenza virus strains A/PR8/8/34 (PR8) and the cold-adapted A/Ann Arbor/6/60 (AA), which are currently used as the donor strains for vaccine seeds due to their attenuated phenotype. We have observed that upon infection with these strains, degradation of the RNAP II does not occur. Moreover, by runoff experiments we observe that PR8 has a reduced ability to inhibit cellular mRNA transcription. In addition, a hypervirulent PR8 (hvPR8) variant that multiplies much faster than standard PR8 (lvPR8) in infected cells and is more virulent in mice than the parental PR8 virus, efficiently induces RNAP II degradation. Studies with reassortant viruses containing defined genome segments of both hvPR8 and lvPR8 indicate that PA and PB2 subunits individually contribute to the ability of influenza virus to degrade the RNAP II. In addition, recently it has been reported that the inclusion of PA or PB2 from hvPR8 in lvPR8 recombinant viruses, highly increases their pathogenicity. Together, the data indicate that the capacity of the influenza virus to degrade RNAP II and inhibit the host cell transcription machinery is a feature of influenza A viruses that might contribute to their virulence.

BLOCKING GENOME GATEKEEPERS

A FAMILY OF ENZYMES KNOWN as histone deacetykses (HDACs) helps to regulate how and when our genome blueprint is transcribed and translated into protein. Small-molecule inhibitors of these enzymes are under intense investigation as cancer therapeutics, as evidenced by research presented last month in Honolulu at the 5th International Chemical Congress of Pacific Basin Societies (Pacifichem). Scientists described their efforts to create HDAC inhibitors for use as both drugs and probes of HDAC biology. Histone deacetylases remove acetyl groups from the histone-packing proteins around which genomic DNA is wrapped in the cell. The acetylation state of histones' lysine side chains helps orchestrate gene transcription: When acetylated, DNA wrapped histones unravel to give the cell's transcription machinery access to the DNA. Acetylation also acts to recruit this machinery When deacetylated, however, histones and DNA pack tightly together to prevent transcription. HDAC inhibitors thus trigger an increase in gene ...

B-MYB, a transcription factor implicated in regulating cell cycle, apoptosis and cancer

B-MYB belongs to the MYB family of transcription factors that include A-MYB and c-MYB. While A-MYB and c-MYB are tissue-specific, B-MYB is broadly expressed in rapidly dividing cells of developing or adult mammals. B-MYBs liaisons with important players of the cell cycle and transcription machinery, such as E2F and retinoblastoma proteins, suggest that its essential function in stem cell formation and mammalian development could be related to its ability to directly or indirectly impinge on gene expression. Besides its role in the cell cycle, B-MYB has been shown to promote cell survival by activating antiapoptotic genes such as ApoJ/clusterin and BCL2. Here, we discuss how B-MYB could be implicated in tumourigenesis by regulating gene expression.

Cyclin-dependent kinase inhibitors: Discovery, development and target rationale for different therapeutic applications

Members of the family of cyclin-dependent kinases (CDKs) fulfill important and partly overlapping roles in the regulation of the cell proliferation cycle and the RNA polymerase-II (RNAP-II) transcription cycle. Clinical development of CDK inhibitors as new anticancer agents has been under way for several years and the first signs of potential efficacy in certain indications have recently been reported. Furthermore, there has been a recent resurgence in the clinical entry of new CDK inhibitors for the treatment of cancer. However, there are numerous other therapeutic applications where inhibition of cell proliferation and transcription with pharmacological CDK inhibitors might be useful. Chief amongst these are proliferative disorders in the areas of nephrology, cardiovascular disease and neurodegeneration. Additionally, infectious diseases in which the invading microorganisms themselves possess CDK-like proteins or depend on the host cell proliferation and transcription machinery are attractive targets. The biomedical rationales for some of these applications of CDK inhibitors are discussed here, with emphasis on the CDK specificity of potential pharmacological agents. Furthermore, progress in the discovery and development of CDK-inhibitory agents is brought up to date.

Abnormal PcG protein expression in Hodgkin's lymphoma. Relation with E2F6 and NFkappaB transcription factors.

The Polycomb group (PcG) of proteins comprises a family of repressors of homeobox genes that play key roles in body formation, haematopoiesis and cell cycle control. In this study, a large-scale analysis of PcG protein expression (BMI1, MEL18, PH1, RNF2, RING1, and RYBP) was performed in 321 Hodgkin's lymphoma (HL) biopsies and in reactive lymphoid tissues using tissue microarrays. The relevance of PcG proteins in HL was also investigated by the simultaneous analysis of PcG and other proteins involved in the control of cell cycle, transcription machinery and lymphoid differentiation. The analysis revealed increased expression of a set of PcG proteins (particularly RYBP and BMI1) in tumour cells in comparison with reactive lymphoid tissue. One of the most striking findings was anomalous RYBP expression in 55% of classical HL cases associated with an unfavourable response to treatment and shorter survival. The data obtained in this study also show an association of PcG proteins with E2F6 and NFkappaB transcription factors. The statistical relationship between PcG and NFkappaB activation was further explored in HL-derived cell lines treated with curcumin, an NFkappaB inhibitor, and TNFalpha. Up- or downregulation of MEL18 was paralleled by loss or gain of activated NFkappaB, which suggests that NFkappaB may regulate expression of this protein. Investigation of the relationship between E2F6 and RING1 by immunofluorescence and confocal analysis, in HL cell lines and paraffin sections, revealed co-expression of both proteins in the same tumour cells. These results allow us to propose that the formation of transcription complexes with E2F6 may modify the functional status of PcG proteins in HSR cells.

CDNA microarray analysis of early gene expression profiles associated with hepatitis B virus X protein-mediated hepatocarcinogenesis

Chronic hepatitis B virus (HBV) infection is one of the major causes of hepatocellular carcinoma. HBV encodes an oncogenic hepatitis B virus X protein (HBx), which can transactivate host cell transcriptional machinery and mediate cellular transformation. To disclose the early genetic response in HBx-mediated transformation process, we constructed a conditional HBx-expressing hepatocyte cell line, which allows us to compare the gene expression profiles under controllable HBx induction. A cDNA microarray containing more than 8700 mouse genes and ESTs was utilized to examine the gene expression profiles. We identified 260 candidate genes and 259 ESTs which have shown aberrant expression under HBx induction. Most of them are involved in signal transduction pathway, cell cycle control, metastasis, transcriptional regulation, immune response, and metabolism. These results provide additional insight into early cellular targets of HBx, which could give us a better understanding of the function of HBx and their progressive changes during HBx-mediated hepatocarcinogenesis.

Global Expression Profiling of Acetate-grown Escherichia coli

This study characterized the transcript profile of Escherichia coli in acetate cultures using DNA microarray on glass slides. Glucose-grown cultures were used as a reference. At the 95% confidence level, 354 genes were up-regulated in acetate, while 370 genes were down-regulated compared with the glucose-grown culture. Generally, more metabolic genes were up-regulated in acetate than other gene groups, while genes involved in cell replication, transcription, and translation machinery tended to be down-regulated. It appears that E. coli commits more resources to metabolism at the expense of growth when cultured in the poor carbon source. The expression profile confirms many known features in acetate metabolism such as the induction of the glyoxylate pathway, tricarboxylic acid cycle, and gluconeogenic genes. It also provided many previously unknown features, including induction of malic enzymes, ppsA, and the glycolate pathway and repression of glycolytic and glucose phosphotransferase genes in acetate. The carbon flux delivered from the malic enzymes and PpsA in acetate was further confirmed by deletion mutations. In general, the gene expression profiles qualitatively agree with the metabolic flux changes and may serve as a predictor for gene function and metabolic flux distribution.

Adenoviral E1A primes alveolar epithelial cells to PM(10)-induced transcription of interleukin-8.

The presence of the adenoviral early region 1A (E1A) protein in human lungs has been associated with an increased risk of chronic obstructive pulmonary disease (COPD), possibly by a mechanism involving amplification of proinflammatory responses. We hypothesize that enhanced inflammation results from increased transcription factor activation in E1A-carrying cells, which may afford susceptibility to environmental particulate matter < 10 microm (PM(10))-mediated oxidative stress. We measured interleukin (IL)-8 mRNA expression and protein release in human alveolar epithelial cells (A549) transfected with the E1A gene (E1A+ve). Both E1A+ve and -ve cells released IL-8 after incubation with TNF-alpha, but only E1A+ve cells were sensitive to LPS stimulation in IL-8 mRNA expression and protein release. E1A+ve cells showed an enhanced IL-8 mRNA and protein response after treatment with H(2)O(2) and PM(10). E1A-enhanced induction of IL-8 was accompanied by increases in activator protein-1 and nuclear factor-kappa B nuclear binding in E1A+ve cells, which also showed higher basal nuclear binding of these transcription factors. These data suggest that the presence of E1A primes the cell transcriptional machinery for oxidative stress signaling and therefore facilitates amplification of proinflammatory responses. By this mechanism, susceptibility to exacerbation of COPD in response to particulate air pollution may occur in individuals harboring E1A.