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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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Treatment of HepG2 cells with 2′AP increased expression of virally encoded E1a, fiber and GFP.(A) HepG2 or (B) MRC5 cells were mock infected or infected with the indicated viruses at MOI of 100 VP/cell and then incubated for 2 days or 4 days, with or without 2.5 mM 2′AP treatment. Cells were washed and lysed and western blot analysis was performed on 10 µg protein per lane using antibodies against E1a, fiber and β-actin. (C) HepG2 and MRC5 cells were infected with AdΔE1b, AdΔE1bΔVA or AdControl at MOI of 100 VP/cell and then incubated in the absence or presence of 2.5 mM 2′AP. Green fluorescence intensity was measured 2 and 4 days post-infection and normalized to uninfected controls. Error bars correspond to +/−SD of quadruplicates (NS – Not Significant; ***p<0.001, one-way ANOVA).
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pone-0065222-g005: Treatment of HepG2 cells with 2′AP increased expression of virally encoded E1a, fiber and GFP.(A) HepG2 or (B) MRC5 cells were mock infected or infected with the indicated viruses at MOI of 100 VP/cell and then incubated for 2 days or 4 days, with or without 2.5 mM 2′AP treatment. Cells were washed and lysed and western blot analysis was performed on 10 µg protein per lane using antibodies against E1a, fiber and β-actin. (C) HepG2 and MRC5 cells were infected with AdΔE1b, AdΔE1bΔVA or AdControl at MOI of 100 VP/cell and then incubated in the absence or presence of 2.5 mM 2′AP. Green fluorescence intensity was measured 2 and 4 days post-infection and normalized to uninfected controls. Error bars correspond to +/−SD of quadruplicates (NS – Not Significant; ***p<0.001, one-way ANOVA).

Mentions: Two days after infection of untreated HepG2 cells with the E1b-fully-deleted viruses, E1a expression (under the control of the mCMV promoter) was similar to that in infections with Ad-dl309, Ad-dl309ΔVA and Ad-dl1520 viruses (under the control of the viral E1a promoter) (Figure 5A, left panel). By 4 days, much higher E1a expression levels were detected in HepG2 cells infected with the E1b-deleted viruses than in cells infected with the other replicating viruses (Figure 5A, right panel). This increase may be due to the loss of E1a autoregulation [58], as a consequence of replacement of the E1a promoter by the mCMV promoter, and/or due to activation of the mCMV promoter by E1a [59]–[61].


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)

Treatment of HepG2 cells with 2′AP increased expression of virally encoded E1a, fiber and GFP.(A) HepG2 or (B) MRC5 cells were mock infected or infected with the indicated viruses at MOI of 100 VP/cell and then incubated for 2 days or 4 days, with or without 2.5 mM 2′AP treatment. Cells were washed and lysed and western blot analysis was performed on 10 µg protein per lane using antibodies against E1a, fiber and β-actin. (C) HepG2 and MRC5 cells were infected with AdΔE1b, AdΔE1bΔVA or AdControl at MOI of 100 VP/cell and then incubated in the absence or presence of 2.5 mM 2′AP. Green fluorescence intensity was measured 2 and 4 days post-infection and normalized to uninfected controls. Error bars correspond to +/−SD of quadruplicates (NS – Not Significant; ***p<0.001, one-way ANOVA).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672087&req=5

pone-0065222-g005: Treatment of HepG2 cells with 2′AP increased expression of virally encoded E1a, fiber and GFP.(A) HepG2 or (B) MRC5 cells were mock infected or infected with the indicated viruses at MOI of 100 VP/cell and then incubated for 2 days or 4 days, with or without 2.5 mM 2′AP treatment. Cells were washed and lysed and western blot analysis was performed on 10 µg protein per lane using antibodies against E1a, fiber and β-actin. (C) HepG2 and MRC5 cells were infected with AdΔE1b, AdΔE1bΔVA or AdControl at MOI of 100 VP/cell and then incubated in the absence or presence of 2.5 mM 2′AP. Green fluorescence intensity was measured 2 and 4 days post-infection and normalized to uninfected controls. Error bars correspond to +/−SD of quadruplicates (NS – Not Significant; ***p<0.001, one-way ANOVA).
Mentions: Two days after infection of untreated HepG2 cells with the E1b-fully-deleted viruses, E1a expression (under the control of the mCMV promoter) was similar to that in infections with Ad-dl309, Ad-dl309ΔVA and Ad-dl1520 viruses (under the control of the viral E1a promoter) (Figure 5A, left panel). By 4 days, much higher E1a expression levels were detected in HepG2 cells infected with the E1b-deleted viruses than in cells infected with the other replicating viruses (Figure 5A, right panel). This increase may be due to the loss of E1a autoregulation [58], as a consequence of replacement of the E1a promoter by the mCMV promoter, and/or due to activation of the mCMV promoter by E1a [59]–[61].

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