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Induction of neuronal and tumor-related genes by adenovirus type 12 E1A.

ABSTRACT: Adenovirus type 12 (Ad12) E1A protein (E1A-12) contains a unique 20-amino-acid spacer region between the second and third conserved regions. Substitution of a single amino acid in the spacer is able to abrogate Ad12 tumorigenesis. To investigate the function of the spacer, microarray analysis was performed on cells transformed by tumorigenic and nontumorigenic Ad12s that differ only by one amino acid in the spacer. Fewer than 0.8% of approximately 8,000 genes in the microarray exhibited differential expression of threefold and higher. Of these, more than half of the known genes with higher expression in the wild-type Ad12-transformed cells have neuronal-specific functions. Some of the other differentially expressed genes are involved in the regulation of the cell cycle, transcription, cell structure, and tumor invasiveness. Northern blot analyses of a subset of the neuronal genes, including Robo1, N-MYC, and alpha-internexin, confirmed their strong expression in multiple Ad12 tumorigenic cell lines. In contrast, these neuronal genes displayed only minor or negligible expression in cells transformed by spacer-mutated Ad12. Significantly, stable introduction of E1A-12 into nontumorigenic Ad5-transformed cells induced neuronal gene expression. We found that the neuron-restrictive silencer factor, which serves as a master repressor of neuronal genes, was inactivated in both Ad12- and Ad5-transformed cells via cytoplasmic retention, though only Ad12-transformed cells exhibited neuronal gene induction. Mutational analyses of the alpha-internexin promoter demonstrated that E1A-12-mediated neuronal gene induction further required the activation of neuronal promoter E-box elements. These results indicate that the spacer is involved in mediating neuronal and tumor-related genes.

SUBMITTER: Guan H

PROVIDER: S-EPMC2612353 | biostudies-literature | 2009 Jan

REPOSITORIES: biostudies-literature

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Induction of neuronal and tumor-related genes by adenovirus type 12 E1A.

Guan Hancheng H Williams Jim F JF Ricciardi Robert P RP

Journal of virology 20081105 2

Adenovirus type 12 (Ad12) E1A protein (E1A-12) contains a unique 20-amino-acid spacer region between the second and third conserved regions. Substitution of a single amino acid in the spacer is able to abrogate Ad12 tumorigenesis. To investigate the function of the spacer, microarray analysis was performed on cells transformed by tumorigenic and nontumorigenic Ad12s that differ only by one amino acid in the spacer. Fewer than 0.8% of approximately 8,000 genes in the microarray exhibited differen ...[more]

PMID: 18987153

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Project description:BackgroundMutations or truncation of the ligand-binding domain (LBD) of androgen receptor (AR) underlie treatment resistance for prostate cancer (PCa). Thus, targeting the AR N-terminal domain (NTD) could overcome such resistance.MethodsLuciferase reporter assays after transient transfection of various DNA constructs were used to assess effects of E1A proteins on AR-mediated transcription. Immunofluorescence microscopy and subcellular fractionation were applied to assess intracellular protein localization. Immunoprecipitation and mammalian two-hybrid assays were used to detect protein-protein interactions. qRT-PCR was employed to determine RNA levels. Western blotting was used to detect protein expression in cells. Effects of adenoviruses on prostate cancer cell survival were evaluated with CellTiter-Glo assays.ResultsAdenovirus 12 E1A (E1A12) binds specifically to the AR. Interestingly, the full-length E1A12 (266 aa) preferentially binds to full-length AR, while the small E1A12 variant (235 aa) interacts more strongly with AR-V7. E1A12 promotes AR nuclear translocation, likely through mediating intramolecular AR NTD-LBD interactions. In the nucleus, AR and E1A12 co-expression in AR-null PCa cells results in E1A12 redistribution from nuclear foci containing CBX4 (also known as Pc2), suggesting a preferential AR-E1A12 interaction over other E1A12 interactors. E1A12 represses AR-mediated transcription in reporter gene assays and endogenous AR target genes such as ATAD2 and MYC in AR-expressing PCa cells. AR-expressing PCa cells are more sensitive to death induced by a recombinant adenovirus expressing E1A12 (Ad-E1A12) than AR-deficient PCa cells, which could be attributed to the increased viral replication promoted by androgen stimulation. Targeting the AR by E1A12 promotes apoptosis in PCa cells that express the full-length AR or C-terminally truncated AR variants. Importantly, inhibition of mTOR signaling that blocks the expression of anti-apoptotic proteins markedly augments Ad-E1A12-induced apoptosis of AR-expressing cells. Mechanistically, Ad-E1A12 infection triggers apoptotic response while activating the PI3K-AKT-mTOR signaling axis; thus, mTOR inhibition enhances apoptosis in AR-expressing PCa cells infected by Ad-E1A12.ConclusionAd12 E1A inhibits AR-mediated transcription and suppresses PCa cell survival, suggesting that targeting the AR by E1A12 might have therapeutic potential for treating advanced PCa with heightened AR signaling.

Project description:The protein products of the adenoviral E1A gene are implicated in a variety of transcriptional and cell cycle events, involving interactions with several proteins present in human cells, including parts of the transcriptional machinery and negative regulators of cell division such as the Rb gene product and p107. To determine if there are functional homologs of E1A in Saccharomyces cerevisiae, we have developed a genetic screen for mutants that depend on E1A for growth. The screen is based on a colony color sectoring assay which allows the identification of mutants dependent on the maintenance and expression of an E1A-containing plasmid. Using this screen, we have isolated five mutants that depend on expression of the 12S or 13S cDNA of E1A for growth. A plasmid shuffle assay confirms that the plasmid-dependent phenotype is due to the presence of either the 12S or the 13S E1A cDNA and that both forms of E1A rescue growth of all mutants equally well. The five mutants fall into two classes that were named web1 and web2 (for "wants E1A badly"). Plasmid shuffle assays with mutant forms of E1A show that conserved region 1 (CR1) is required for rescue of the growth of the web1 and web2 E1A-dependent yeast mutants, while the N-terminal 22 amino acids are only partially required; conserved region 2 (CR2) and the C terminus are dispensable. The phenotypes of mutants in both the web1 and the web2 groups are due to a single gene defect, and the yeast genes that fully complement the mutant phenotypes of both groups were cloned. The WEB1 gene sequence encodes a 1,273-amino-acid protein that is identical to SEC31, a protein involved in the budding of transport vesicles from the endoplasmic reticulum. The WEB2 gene encodes a 1,522-amino-acid protein with homology to nucleic acid-dependent ATPases. Deletion of either WEB1 or WEB2 is lethal. Expression of E1A is not able to rescue the lethality of either the web1 or the web2 null allele, implying allele-specific mutations that lead to E1A dependence.

Dataset Information

Induction of neuronal and tumor-related genes by adenovirus type 12 E1A.

Publications

Induction of neuronal and tumor-related genes by adenovirus type 12 E1A.

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