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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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2′AP treatment increased both Ad-dl309 and AdΔE1b mediated HepG2 cell death.(A) parental HepG2, (B) HepG2-E1b-Mut and (C, D) HepG2-E1b-WT cells were infected with indicated viruses at MOI of 10 (D) or 100 (A, B, C) VP/cell and cultured in medium with or without 2.5 mM 2′AP. Survival was determined using an Alamar Blue assay 6 days post-infection. Fluorescence measurements were normalized to AdControl infected wells. Error bars correspond to +/−SD of quadruplicates (NS – Not Significant; ***p<0.001, **p<0.01, *p<0.05, one-way ANOVA).
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pone-0065222-g009: 2′AP treatment increased both Ad-dl309 and AdΔE1b mediated HepG2 cell death.(A) parental HepG2, (B) HepG2-E1b-Mut and (C, D) HepG2-E1b-WT cells were infected with indicated viruses at MOI of 10 (D) or 100 (A, B, C) VP/cell and cultured in medium with or without 2.5 mM 2′AP. Survival was determined using an Alamar Blue assay 6 days post-infection. Fluorescence measurements were normalized to AdControl infected wells. Error bars correspond to +/−SD of quadruplicates (NS – Not Significant; ***p<0.001, **p<0.01, *p<0.05, one-way ANOVA).

Mentions: Since 2′AP treatment and E1b-55K expression increased E1b-deleted virus production in a complementary manner, we investigated whether complementation would also be observed with virus-mediated cell death. HepG2-E1b-WT, HepG2-E1b-Mut and parental HepG2 cells were infected with the replicating viruses and incubated 6 days with or without 2.5 mM 2′AP prior to survival assessment (Figure 9). In the absence of 2′AP, efficient killing by virus at an MOI of 100 VP/cell (about 1 infectious unit/cell) required an intact E1b-55K protein either encoded by the virus (e.g., Ad-dl309 in Figure 9A) or provided in trans by the cells (e.g., Figure 9C). An activity of E1b-55K that is dependent on cysteine residues at 454 and/or 456 appeared to be critical for virus-mediated cell death, as the E1b-55K mutant protein did not complement the E1b-deleted viruses (Figure 9B). Furthermore, similar to its effect on virus production (Figure 6), 2′AP treatment could substitute for E1b-55K in killing by AdΔE1b and AdΔE1bΔVA (Figure 9 A, B). In contrast to AdΔE1b, Ad-dl1520 did not induce greater killing of these cells when combined with 2′AP treatment, which may be due to lower E1a levels in 2′AP-treated cells infected with Ad-dl1520 compared to AdΔE1b.


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)

2′AP treatment increased both Ad-dl309 and AdΔE1b mediated HepG2 cell death.(A) parental HepG2, (B) HepG2-E1b-Mut and (C, D) HepG2-E1b-WT cells were infected with indicated viruses at MOI of 10 (D) or 100 (A, B, C) VP/cell and cultured in medium with or without 2.5 mM 2′AP. Survival was determined using an Alamar Blue assay 6 days post-infection. Fluorescence measurements were normalized to AdControl infected wells. Error bars correspond to +/−SD of quadruplicates (NS – Not Significant; ***p<0.001, **p<0.01, *p<0.05, one-way ANOVA).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672087&req=5

pone-0065222-g009: 2′AP treatment increased both Ad-dl309 and AdΔE1b mediated HepG2 cell death.(A) parental HepG2, (B) HepG2-E1b-Mut and (C, D) HepG2-E1b-WT cells were infected with indicated viruses at MOI of 10 (D) or 100 (A, B, C) VP/cell and cultured in medium with or without 2.5 mM 2′AP. Survival was determined using an Alamar Blue assay 6 days post-infection. Fluorescence measurements were normalized to AdControl infected wells. Error bars correspond to +/−SD of quadruplicates (NS – Not Significant; ***p<0.001, **p<0.01, *p<0.05, one-way ANOVA).
Mentions: Since 2′AP treatment and E1b-55K expression increased E1b-deleted virus production in a complementary manner, we investigated whether complementation would also be observed with virus-mediated cell death. HepG2-E1b-WT, HepG2-E1b-Mut and parental HepG2 cells were infected with the replicating viruses and incubated 6 days with or without 2.5 mM 2′AP prior to survival assessment (Figure 9). In the absence of 2′AP, efficient killing by virus at an MOI of 100 VP/cell (about 1 infectious unit/cell) required an intact E1b-55K protein either encoded by the virus (e.g., Ad-dl309 in Figure 9A) or provided in trans by the cells (e.g., Figure 9C). An activity of E1b-55K that is dependent on cysteine residues at 454 and/or 456 appeared to be critical for virus-mediated cell death, as the E1b-55K mutant protein did not complement the E1b-deleted viruses (Figure 9B). Furthermore, similar to its effect on virus production (Figure 6), 2′AP treatment could substitute for E1b-55K in killing by AdΔE1b and AdΔE1bΔVA (Figure 9 A, B). In contrast to AdΔE1b, Ad-dl1520 did not induce greater killing of these cells when combined with 2′AP treatment, which may be due to lower E1a levels in 2′AP-treated cells infected with Ad-dl1520 compared to AdΔE1b.

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