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2-aminopurine enhances the oncolytic activity of an E1b-deleted adenovirus in hepatocellular carcinoma cells.

Sharon D, Schümann M, MacLeod S, McPherson R, Chaurasiya S, Shaw A, Hitt MM - PLoS ONE (2013)

Bottom Line: Furthermore, these mutations were also found to reduce the activity of the replicating viruses in certain cancer cells.We constructed an adenovirus with deletions of both the E1b and the VA-RNA genes and found that replication of this virus was selective for human hepatocellular carcinoma (HCC) cell lines when compared to normal cell lines.Furthermore, we show that 2-aminopurine (2'AP) treatment selectively enhanced virus replication and virus-mediated death of HCC cells. 2'AP did not compensate for the loss of VA-RNA activities, but rather the loss of an E1b-55K activity, such as the DNA damage response, suggesting that co-administration of 2'AP derivatives that block host DNA damage response, may increase the oncolytic activity of AdΔE1bΔVA without reducing its selectivity for HCC cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, University of Alberta, Edmonton, Alberta, Canada.

ABSTRACT
Adenoviruses with deletions of viral genes have been extensively studied as potential cancer therapeutics. Although a high degree of cancer selectivity has been demonstrated with these conditionally replicating adenoviruses, low levels of virus replication can be detected in normal cells. Furthermore, these mutations were also found to reduce the activity of the replicating viruses in certain cancer cells. Recent studies have shown that co-administration of chemotherapeutic drugs may increase the activity of these viruses without affecting their specificity. We constructed an adenovirus with deletions of both the E1b and the VA-RNA genes and found that replication of this virus was selective for human hepatocellular carcinoma (HCC) cell lines when compared to normal cell lines. Furthermore, we show that 2-aminopurine (2'AP) treatment selectively enhanced virus replication and virus-mediated death of HCC cells. 2'AP did not compensate for the loss of VA-RNA activities, but rather the loss of an E1b-55K activity, such as the DNA damage response, suggesting that co-administration of 2'AP derivatives that block host DNA damage response, may increase the oncolytic activity of AdΔE1bΔVA without reducing its selectivity for HCC cells.

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p53 expression and activity were not inhibited by 2′AP treatment of infected cells.(A) HepG2 and Hep3B cells were infected with Ad-dl309, Ad-dl1520 or AdΔE1b at MOI of 100 VP/cell and treated with no drug or 2.5 mM 2′AP for 2 days. p53 and E1a levels were detected by western blot analysis. (B) HepG2 and Hep3B cells were transfected with a p53-responsive firefly luciferase expression vector. The next day, transfected cells were mock infected or infected with Ad-dl309, Ad-dl1520 or AdΔE1b at MOI of 100 VP/cell and treated with no drug or 2.5 mM 2′AP. Luciferase expression was measured 2 days post-infection. Both HepG2 and Hep3B have high transfection efficiencies. Error bars correspond to +/−SD of triplicates.
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pone-0065222-g007: p53 expression and activity were not inhibited by 2′AP treatment of infected cells.(A) HepG2 and Hep3B cells were infected with Ad-dl309, Ad-dl1520 or AdΔE1b at MOI of 100 VP/cell and treated with no drug or 2.5 mM 2′AP for 2 days. p53 and E1a levels were detected by western blot analysis. (B) HepG2 and Hep3B cells were transfected with a p53-responsive firefly luciferase expression vector. The next day, transfected cells were mock infected or infected with Ad-dl309, Ad-dl1520 or AdΔE1b at MOI of 100 VP/cell and treated with no drug or 2.5 mM 2′AP. Luciferase expression was measured 2 days post-infection. Both HepG2 and Hep3B have high transfection efficiencies. Error bars correspond to +/−SD of triplicates.

Mentions: One of the main functions of E1b-55K in an infected cell is to block p53 pathways, either by inhibiting p53 activity or decreasing its stability [2]–[4]. Here we examined the potential influence of p53 on replication of E1b-deleted virus in p53-positive HepG2 [62]. In HepG2 cells, p53 was only detectable in infections with AdΔE1b (Figure 7A). This is consistent with E1a-mediated stabilization of p53 [63]–[66]. However, we did not detect p53 in Ad-dl1520-infected HepG2 cells which had levels of E1a comparable to AdΔE1b infected cells at this time point. As Ad-dl1520 has previously been shown to induce or stabilize p53 levels [9], [67], [68], it is not immediately apparent why this virus did not enhance p53 protein levels in our system.


2-aminopurine enhances the oncolytic activity of an E1b-deleted adenovirus in hepatocellular carcinoma cells.

Sharon D, Schümann M, MacLeod S, McPherson R, Chaurasiya S, Shaw A, Hitt MM - PLoS ONE (2013)

p53 expression and activity were not inhibited by 2′AP treatment of infected cells.(A) HepG2 and Hep3B cells were infected with Ad-dl309, Ad-dl1520 or AdΔE1b at MOI of 100 VP/cell and treated with no drug or 2.5 mM 2′AP for 2 days. p53 and E1a levels were detected by western blot analysis. (B) HepG2 and Hep3B cells were transfected with a p53-responsive firefly luciferase expression vector. The next day, transfected cells were mock infected or infected with Ad-dl309, Ad-dl1520 or AdΔE1b at MOI of 100 VP/cell and treated with no drug or 2.5 mM 2′AP. Luciferase expression was measured 2 days post-infection. Both HepG2 and Hep3B have high transfection efficiencies. Error bars correspond to +/−SD of triplicates.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672087&req=5

pone-0065222-g007: p53 expression and activity were not inhibited by 2′AP treatment of infected cells.(A) HepG2 and Hep3B cells were infected with Ad-dl309, Ad-dl1520 or AdΔE1b at MOI of 100 VP/cell and treated with no drug or 2.5 mM 2′AP for 2 days. p53 and E1a levels were detected by western blot analysis. (B) HepG2 and Hep3B cells were transfected with a p53-responsive firefly luciferase expression vector. The next day, transfected cells were mock infected or infected with Ad-dl309, Ad-dl1520 or AdΔE1b at MOI of 100 VP/cell and treated with no drug or 2.5 mM 2′AP. Luciferase expression was measured 2 days post-infection. Both HepG2 and Hep3B have high transfection efficiencies. Error bars correspond to +/−SD of triplicates.
Mentions: One of the main functions of E1b-55K in an infected cell is to block p53 pathways, either by inhibiting p53 activity or decreasing its stability [2]–[4]. Here we examined the potential influence of p53 on replication of E1b-deleted virus in p53-positive HepG2 [62]. In HepG2 cells, p53 was only detectable in infections with AdΔE1b (Figure 7A). This is consistent with E1a-mediated stabilization of p53 [63]–[66]. However, we did not detect p53 in Ad-dl1520-infected HepG2 cells which had levels of E1a comparable to AdΔE1b infected cells at this time point. As Ad-dl1520 has previously been shown to induce or stabilize p53 levels [9], [67], [68], it is not immediately apparent why this virus did not enhance p53 protein levels in our system.

Bottom Line: Furthermore, these mutations were also found to reduce the activity of the replicating viruses in certain cancer cells.We constructed an adenovirus with deletions of both the E1b and the VA-RNA genes and found that replication of this virus was selective for human hepatocellular carcinoma (HCC) cell lines when compared to normal cell lines.Furthermore, we show that 2-aminopurine (2'AP) treatment selectively enhanced virus replication and virus-mediated death of HCC cells. 2'AP did not compensate for the loss of VA-RNA activities, but rather the loss of an E1b-55K activity, such as the DNA damage response, suggesting that co-administration of 2'AP derivatives that block host DNA damage response, may increase the oncolytic activity of AdΔE1bΔVA without reducing its selectivity for HCC cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, University of Alberta, Edmonton, Alberta, Canada.

ABSTRACT
Adenoviruses with deletions of viral genes have been extensively studied as potential cancer therapeutics. Although a high degree of cancer selectivity has been demonstrated with these conditionally replicating adenoviruses, low levels of virus replication can be detected in normal cells. Furthermore, these mutations were also found to reduce the activity of the replicating viruses in certain cancer cells. Recent studies have shown that co-administration of chemotherapeutic drugs may increase the activity of these viruses without affecting their specificity. We constructed an adenovirus with deletions of both the E1b and the VA-RNA genes and found that replication of this virus was selective for human hepatocellular carcinoma (HCC) cell lines when compared to normal cell lines. Furthermore, we show that 2-aminopurine (2'AP) treatment selectively enhanced virus replication and virus-mediated death of HCC cells. 2'AP did not compensate for the loss of VA-RNA activities, but rather the loss of an E1b-55K activity, such as the DNA damage response, suggesting that co-administration of 2'AP derivatives that block host DNA damage response, may increase the oncolytic activity of AdΔE1bΔVA without reducing its selectivity for HCC cells.

Show MeSH
Related in: MedlinePlus