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Identification of genes differentially expressed as result of adenovirus type 5- and adenovirus type 12-transformation.

Strath J, Georgopoulos LJ, Kellam P, Blair GE - BMC Genomics (2009)

Bottom Line: Analysis of microarray data revealed that a total of 232 genes were differentially expressed in Ad12 E1- or Ad5 E1-transformed BRK cells compared to untransformed baby rat kidney (BRK) cells.Up-regulation of RelA and significant dysregulation of collagen type I mRNA transcripts and proteins were found in Ad-transformed cells.These results suggest that a complex web of cellular pathways become altered in Ad-transformed cells and that Ad E1A is sufficient for the observed dysregulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK. janet_strath@hotmail.com

ABSTRACT

Background: Cells transformed by human adenoviruses (Ad) exhibit differential capacities to induce tumours in immunocompetent rodents; for example, Ad12-transformed rodent cells are oncogenic whereas Ad5-transformed cells are not. The E1A gene determines oncogenic phenotype, is a transcriptional regulator and dysregulates host cell gene expression, a key factor in both cellular transformation and oncogenesis. To reveal differences in gene expression between cells transformed with oncogenic and non-oncogenic adenoviruses we have performed comparative analysis of transcript profiles with the aim of identifying candidate genes involved in the process of neoplastic transformation.

Results: Analysis of microarray data revealed that a total of 232 genes were differentially expressed in Ad12 E1- or Ad5 E1-transformed BRK cells compared to untransformed baby rat kidney (BRK) cells. Gene information was available for 193 transcripts and using gene ontology (GO) classifications and literature searches it was possible to assign known or suggested functions to 166 of these identified genes. A subset of differentially-expressed genes from the microarray was further examined by real-time PCR and Western blotting using BRK cells immortalised by Ad12 E1A or Ad5 E1A in addition to Ad12 E1- or Ad5 E1-transformed BRK cells. Up-regulation of RelA and significant dysregulation of collagen type I mRNA transcripts and proteins were found in Ad-transformed cells.

Conclusion: These results suggest that a complex web of cellular pathways become altered in Ad-transformed cells and that Ad E1A is sufficient for the observed dysregulation. Further work will focus on investigating which splice variant of Ad E1A is responsible for the observed dysregulation at the pathway level, and the mechanisms of E1A-mediated transcriptional regulation.

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Microarray results and corresponding RT-PCR results for ZBTB22 and NFAT5 expression in Ad-transformed cells. The microarray results for ZBTB22 indicating up-regulation of the transcript in Ad12 E1-TC were validated by real-time PCR. The microarray result for NFAT5 indicating significant up-regulation in Ad5 E1-TC (1.58-fold; table 1) was validated by real-time PCR. The down-regulation in Ad12 E1-TC indicated by the microarray study was not significant (0.17-fold; table 1) due to high variability (p-value; 0.635) and the real-time PCR results for expression of NFAT5 also did not indicate any significant dysregulation of this transcript in Ad12 E1-TC, or Ad12-TC.
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Figure 11: Microarray results and corresponding RT-PCR results for ZBTB22 and NFAT5 expression in Ad-transformed cells. The microarray results for ZBTB22 indicating up-regulation of the transcript in Ad12 E1-TC were validated by real-time PCR. The microarray result for NFAT5 indicating significant up-regulation in Ad5 E1-TC (1.58-fold; table 1) was validated by real-time PCR. The down-regulation in Ad12 E1-TC indicated by the microarray study was not significant (0.17-fold; table 1) due to high variability (p-value; 0.635) and the real-time PCR results for expression of NFAT5 also did not indicate any significant dysregulation of this transcript in Ad12 E1-TC, or Ad12-TC.

Mentions: Other targets that were not mapped by IPA but were identified by manual BLAST searching include ZBTB22, NFAT5 and SSG1. No GO information about biological function is available for ZBTB22 (synonyms ZNF297 and BING1), originally identified as a gene within the 70 kbp segment flanking the TAPASIN locus on human chromosome 6 [56], within the extended collection of genes known as the Major Histocompatibility Complex (MHC). As ZBTB22 contains both a zinc finger domain and a POZ domain, the suspected function is transcriptional repression. Figure 11 shows that there is good agreement between the microarray and real-time PCR results, with up-regulation of ZBTB22 in Ad5 E1-TC and, to a lesser extent, in Ad12 E1-TC compared to untransformed BRK cells. All of the genes assigned to the stress and immune response category were found to be up-regulated in Ad5 E1A-TC compared to either untransformed BRK cells or Ad12 E1A-TC (Figure 2). These genes include NFAT5, which was confirmed by real-time PCR results as being up-regulated in Ad5 E1A-immortalised cells compared to BRK cells (Figure 11).


Identification of genes differentially expressed as result of adenovirus type 5- and adenovirus type 12-transformation.

Strath J, Georgopoulos LJ, Kellam P, Blair GE - BMC Genomics (2009)

Microarray results and corresponding RT-PCR results for ZBTB22 and NFAT5 expression in Ad-transformed cells. The microarray results for ZBTB22 indicating up-regulation of the transcript in Ad12 E1-TC were validated by real-time PCR. The microarray result for NFAT5 indicating significant up-regulation in Ad5 E1-TC (1.58-fold; table 1) was validated by real-time PCR. The down-regulation in Ad12 E1-TC indicated by the microarray study was not significant (0.17-fold; table 1) due to high variability (p-value; 0.635) and the real-time PCR results for expression of NFAT5 also did not indicate any significant dysregulation of this transcript in Ad12 E1-TC, or Ad12-TC.
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Related In: Results  -  Collection

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Figure 11: Microarray results and corresponding RT-PCR results for ZBTB22 and NFAT5 expression in Ad-transformed cells. The microarray results for ZBTB22 indicating up-regulation of the transcript in Ad12 E1-TC were validated by real-time PCR. The microarray result for NFAT5 indicating significant up-regulation in Ad5 E1-TC (1.58-fold; table 1) was validated by real-time PCR. The down-regulation in Ad12 E1-TC indicated by the microarray study was not significant (0.17-fold; table 1) due to high variability (p-value; 0.635) and the real-time PCR results for expression of NFAT5 also did not indicate any significant dysregulation of this transcript in Ad12 E1-TC, or Ad12-TC.
Mentions: Other targets that were not mapped by IPA but were identified by manual BLAST searching include ZBTB22, NFAT5 and SSG1. No GO information about biological function is available for ZBTB22 (synonyms ZNF297 and BING1), originally identified as a gene within the 70 kbp segment flanking the TAPASIN locus on human chromosome 6 [56], within the extended collection of genes known as the Major Histocompatibility Complex (MHC). As ZBTB22 contains both a zinc finger domain and a POZ domain, the suspected function is transcriptional repression. Figure 11 shows that there is good agreement between the microarray and real-time PCR results, with up-regulation of ZBTB22 in Ad5 E1-TC and, to a lesser extent, in Ad12 E1-TC compared to untransformed BRK cells. All of the genes assigned to the stress and immune response category were found to be up-regulated in Ad5 E1A-TC compared to either untransformed BRK cells or Ad12 E1A-TC (Figure 2). These genes include NFAT5, which was confirmed by real-time PCR results as being up-regulated in Ad5 E1A-immortalised cells compared to BRK cells (Figure 11).

Bottom Line: Analysis of microarray data revealed that a total of 232 genes were differentially expressed in Ad12 E1- or Ad5 E1-transformed BRK cells compared to untransformed baby rat kidney (BRK) cells.Up-regulation of RelA and significant dysregulation of collagen type I mRNA transcripts and proteins were found in Ad-transformed cells.These results suggest that a complex web of cellular pathways become altered in Ad-transformed cells and that Ad E1A is sufficient for the observed dysregulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK. janet_strath@hotmail.com

ABSTRACT

Background: Cells transformed by human adenoviruses (Ad) exhibit differential capacities to induce tumours in immunocompetent rodents; for example, Ad12-transformed rodent cells are oncogenic whereas Ad5-transformed cells are not. The E1A gene determines oncogenic phenotype, is a transcriptional regulator and dysregulates host cell gene expression, a key factor in both cellular transformation and oncogenesis. To reveal differences in gene expression between cells transformed with oncogenic and non-oncogenic adenoviruses we have performed comparative analysis of transcript profiles with the aim of identifying candidate genes involved in the process of neoplastic transformation.

Results: Analysis of microarray data revealed that a total of 232 genes were differentially expressed in Ad12 E1- or Ad5 E1-transformed BRK cells compared to untransformed baby rat kidney (BRK) cells. Gene information was available for 193 transcripts and using gene ontology (GO) classifications and literature searches it was possible to assign known or suggested functions to 166 of these identified genes. A subset of differentially-expressed genes from the microarray was further examined by real-time PCR and Western blotting using BRK cells immortalised by Ad12 E1A or Ad5 E1A in addition to Ad12 E1- or Ad5 E1-transformed BRK cells. Up-regulation of RelA and significant dysregulation of collagen type I mRNA transcripts and proteins were found in Ad-transformed cells.

Conclusion: These results suggest that a complex web of cellular pathways become altered in Ad-transformed cells and that Ad E1A is sufficient for the observed dysregulation. Further work will focus on investigating which splice variant of Ad E1A is responsible for the observed dysregulation at the pathway level, and the mechanisms of E1A-mediated transcriptional regulation.

Show MeSH
Related in: MedlinePlus