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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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Relative levels of E1A expression in transformed cell lines. Levels of E1A expression in the transformed cell lines were assessed by Western Blot. Intensities were corrected for average background and normalised to GAPDH expression calculated individually for each loaded sample. The expression levels are given as percentages of the intensity for cells transformed with Ad5 or Ad12 inactivated virus. Results for Ad5 E1A expression are from an average of six membranes and values for Ad12 E1A expression represent the average from seven membranes.
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Figure 5: Relative levels of E1A expression in transformed cell lines. Levels of E1A expression in the transformed cell lines were assessed by Western Blot. Intensities were corrected for average background and normalised to GAPDH expression calculated individually for each loaded sample. The expression levels are given as percentages of the intensity for cells transformed with Ad5 or Ad12 inactivated virus. Results for Ad5 E1A expression are from an average of six membranes and values for Ad12 E1A expression represent the average from seven membranes.

Mentions: From this analysis, six genes indicative of defined biological processes were selected for further study at the mRNA level by real-time PCR; V-rel reticuloendotheliosis viral oncogene homolog A (avian) (RelA), zinc finger and BTB domain containing 22 (ZBTB22), nuclear factor of activated T-cells (NFAT5), collagen type 1 alpha 1 (COL1A1), mixed-lineage kinase 1 (MLK1; synonym MAP3K9) and protein phosphatase regulatory (inhibitor) subunit 1A (PPP1R1A). The expression levels of these mRNA transcripts were investigated in the Ad E1-TCs used for the microarray study and also in BRK cells immortalised with Ad12 (Ad12 E1A-TC) or Ad5 E1A (Ad5 E1A-TC) alone. Expression of COL1A1 was investigated at the protein level by immunofluorescence microscopy and expression of RelA, MLK1 and PPP1R1A was also investigated at the protein level using cell lysates derived from the previously mentioned cell-lines and also from cells transformed with inactivated Ad5 (Ad5-TC) or inactivated Ad12 (Ad12-TC). Relative levels of E1A expression in these cell lines are indicated in figure 5. Differential splicing of the E1A transcript and phosphorylation of the E1A protein gives rise to several bands ranging from approximately 35 to 47 kDa [14]. The cell line transformed with inactivated Ad5 showed the highest level of E1A expression in comparison with the cell lines transformed with Ad5 E1 or E1A alone. This could be related to the number of copies of the viral DNA sequences that have been integrated in each cell line. Previous studies have shown that adenovirus DNA integrates at multiple sites (from around 5 to 30 copies of the viral genome per cell) and these are subject to methylation and loss/rearrangement, such that only the transforming (E1A and E1B) are constitutively expressed in the transformed cell thus driving cell proliferation [15]. Expression of E1A in cell lines immortalised by the AccI-H fragment corresponding to the 4.7% of the Ad12 genome (Ad12 E1A-TC) or the HpaI-E fragment corresponding to the left-terminal 4.5% of the Ad5 genome (Ad5 E1A-TC) is lower than that shown by cell lines transformed by the entire E1 region, in agreement with previous observations that the E1B region acts to increase the frequency and stability of transformation by E1A [16]. The lowest levels of E1A expression are in the Ad5 E1A immortalised cell-lines; probably due to the absence of the poly (A) signal of E1A in the HpaI-E fragment of the Ad5 genome used to immortalise this cell line [17].


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)

Relative levels of E1A expression in transformed cell lines. Levels of E1A expression in the transformed cell lines were assessed by Western Blot. Intensities were corrected for average background and normalised to GAPDH expression calculated individually for each loaded sample. The expression levels are given as percentages of the intensity for cells transformed with Ad5 or Ad12 inactivated virus. Results for Ad5 E1A expression are from an average of six membranes and values for Ad12 E1A expression represent the average from seven membranes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651901&req=5

Figure 5: Relative levels of E1A expression in transformed cell lines. Levels of E1A expression in the transformed cell lines were assessed by Western Blot. Intensities were corrected for average background and normalised to GAPDH expression calculated individually for each loaded sample. The expression levels are given as percentages of the intensity for cells transformed with Ad5 or Ad12 inactivated virus. Results for Ad5 E1A expression are from an average of six membranes and values for Ad12 E1A expression represent the average from seven membranes.
Mentions: From this analysis, six genes indicative of defined biological processes were selected for further study at the mRNA level by real-time PCR; V-rel reticuloendotheliosis viral oncogene homolog A (avian) (RelA), zinc finger and BTB domain containing 22 (ZBTB22), nuclear factor of activated T-cells (NFAT5), collagen type 1 alpha 1 (COL1A1), mixed-lineage kinase 1 (MLK1; synonym MAP3K9) and protein phosphatase regulatory (inhibitor) subunit 1A (PPP1R1A). The expression levels of these mRNA transcripts were investigated in the Ad E1-TCs used for the microarray study and also in BRK cells immortalised with Ad12 (Ad12 E1A-TC) or Ad5 E1A (Ad5 E1A-TC) alone. Expression of COL1A1 was investigated at the protein level by immunofluorescence microscopy and expression of RelA, MLK1 and PPP1R1A was also investigated at the protein level using cell lysates derived from the previously mentioned cell-lines and also from cells transformed with inactivated Ad5 (Ad5-TC) or inactivated Ad12 (Ad12-TC). Relative levels of E1A expression in these cell lines are indicated in figure 5. Differential splicing of the E1A transcript and phosphorylation of the E1A protein gives rise to several bands ranging from approximately 35 to 47 kDa [14]. The cell line transformed with inactivated Ad5 showed the highest level of E1A expression in comparison with the cell lines transformed with Ad5 E1 or E1A alone. This could be related to the number of copies of the viral DNA sequences that have been integrated in each cell line. Previous studies have shown that adenovirus DNA integrates at multiple sites (from around 5 to 30 copies of the viral genome per cell) and these are subject to methylation and loss/rearrangement, such that only the transforming (E1A and E1B) are constitutively expressed in the transformed cell thus driving cell proliferation [15]. Expression of E1A in cell lines immortalised by the AccI-H fragment corresponding to the 4.7% of the Ad12 genome (Ad12 E1A-TC) or the HpaI-E fragment corresponding to the left-terminal 4.5% of the Ad5 genome (Ad5 E1A-TC) is lower than that shown by cell lines transformed by the entire E1 region, in agreement with previous observations that the E1B region acts to increase the frequency and stability of transformation by E1A [16]. The lowest levels of E1A expression are in the Ad5 E1A immortalised cell-lines; probably due to the absence of the poly (A) signal of E1A in the HpaI-E fragment of the Ad5 genome used to immortalise this cell line [17].

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