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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 rescued the C454S/C456S E1b-55K mutation in HepG2 cells.(A) Parental HepG2, HepG2-E1b-WT and HepG2-E1b-Mut were infected with Ad-dl309, Ad-dl1520 or AdΔE1b at MOI of 1 PFU/cell for 1 hour prior to treatment with medium containing no drug or 2.5 mM 2′AP. Infected cells and media were harvested 4 days post-infection and virus yields were determine by plaque assays on HEK293 cells. (B) Cells were infected with AdΔE1b at MOI of 1 GFU/cell for 1 hour prior to treatment with medium containing no drug or 2.5 mM 2′AP. Lysates from infected cells were harvested at 1 hr (day 0) as well as 1, 2, 3 and 4 days post-infection. Virus yields were determined by titration in Hep3B cells as described in the materials and methods. Error bars correspond to +/−SD of quadruplicates (NS – Not Significant; ****p<0.0001, **p<0.01, one-way ANOVA).
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pone-0065222-g008: 2′AP rescued the C454S/C456S E1b-55K mutation in HepG2 cells.(A) Parental HepG2, HepG2-E1b-WT and HepG2-E1b-Mut were infected with Ad-dl309, Ad-dl1520 or AdΔE1b at MOI of 1 PFU/cell for 1 hour prior to treatment with medium containing no drug or 2.5 mM 2′AP. Infected cells and media were harvested 4 days post-infection and virus yields were determine by plaque assays on HEK293 cells. (B) Cells were infected with AdΔE1b at MOI of 1 GFU/cell for 1 hour prior to treatment with medium containing no drug or 2.5 mM 2′AP. Lysates from infected cells were harvested at 1 hr (day 0) as well as 1, 2, 3 and 4 days post-infection. Virus yields were determined by titration in Hep3B cells as described in the materials and methods. Error bars correspond to +/−SD of quadruplicates (NS – Not Significant; ****p<0.0001, **p<0.01, one-way ANOVA).

Mentions: Both 2′AP and E1b-55K have been shown to block the DNA damage response [13], [14], [44], [45]. Furthermore, previous studies have found that one of the E1b-55K domains responsible for this inhibition includes the residues C454 and C456 [17], [18]. Therefore, we tested whether inhibition of the DNA damage response could potentially be involved in E1b-deleted virus production in HepG2 cells treated with 2′AP. We stably transfected HepG2 cells with vectors encoding either wild-type E1b-55K or a mutated E1b-55K (C454S/C456S). The stably transfected HepG2 cells (HepG2-E1b-WT and HepG2-E1b-Mut) as well as the parental HepG2 cells were infected with Ad-dl309, Ad-dl1520 or AdΔE1b then cultured with or without 2′AP treatment for 4 days prior to virus production measurement (Figure 8A). While 2′AP had no effect on Ad-dl309 production in any of these cell lines, 2′AP strongly increased production of both Ad-dl1520 and AdΔE1b in parental HepG2 cells as well as HepG2-E1b-Mut cells. As expected, HepG2-E1b-WT at least partially complemented the E1b deletions in Ad-dl1520 and AdΔE1b allowing relatively high virus production, and 2′AP treatment did not further increase yields substantially. Interestingly, AdΔE1b production levels were much lower than Ad-dl1520 even in the complementing HepG2-E1b-WT cells, suggesting that other differences between Ad-dl1520 and AdΔE1b, such as E1b-19K and the E3 death protein encoded by Ad-dl1520, or mCMV-promoter-control of E1a in AdΔE1b, may also contributed to lower AdΔE1b production.


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 rescued the C454S/C456S E1b-55K mutation in HepG2 cells.(A) Parental HepG2, HepG2-E1b-WT and HepG2-E1b-Mut were infected with Ad-dl309, Ad-dl1520 or AdΔE1b at MOI of 1 PFU/cell for 1 hour prior to treatment with medium containing no drug or 2.5 mM 2′AP. Infected cells and media were harvested 4 days post-infection and virus yields were determine by plaque assays on HEK293 cells. (B) Cells were infected with AdΔE1b at MOI of 1 GFU/cell for 1 hour prior to treatment with medium containing no drug or 2.5 mM 2′AP. Lysates from infected cells were harvested at 1 hr (day 0) as well as 1, 2, 3 and 4 days post-infection. Virus yields were determined by titration in Hep3B cells as described in the materials and methods. Error bars correspond to +/−SD of quadruplicates (NS – Not Significant; ****p<0.0001, **p<0.01, one-way ANOVA).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672087&req=5

pone-0065222-g008: 2′AP rescued the C454S/C456S E1b-55K mutation in HepG2 cells.(A) Parental HepG2, HepG2-E1b-WT and HepG2-E1b-Mut were infected with Ad-dl309, Ad-dl1520 or AdΔE1b at MOI of 1 PFU/cell for 1 hour prior to treatment with medium containing no drug or 2.5 mM 2′AP. Infected cells and media were harvested 4 days post-infection and virus yields were determine by plaque assays on HEK293 cells. (B) Cells were infected with AdΔE1b at MOI of 1 GFU/cell for 1 hour prior to treatment with medium containing no drug or 2.5 mM 2′AP. Lysates from infected cells were harvested at 1 hr (day 0) as well as 1, 2, 3 and 4 days post-infection. Virus yields were determined by titration in Hep3B cells as described in the materials and methods. Error bars correspond to +/−SD of quadruplicates (NS – Not Significant; ****p<0.0001, **p<0.01, one-way ANOVA).
Mentions: Both 2′AP and E1b-55K have been shown to block the DNA damage response [13], [14], [44], [45]. Furthermore, previous studies have found that one of the E1b-55K domains responsible for this inhibition includes the residues C454 and C456 [17], [18]. Therefore, we tested whether inhibition of the DNA damage response could potentially be involved in E1b-deleted virus production in HepG2 cells treated with 2′AP. We stably transfected HepG2 cells with vectors encoding either wild-type E1b-55K or a mutated E1b-55K (C454S/C456S). The stably transfected HepG2 cells (HepG2-E1b-WT and HepG2-E1b-Mut) as well as the parental HepG2 cells were infected with Ad-dl309, Ad-dl1520 or AdΔE1b then cultured with or without 2′AP treatment for 4 days prior to virus production measurement (Figure 8A). While 2′AP had no effect on Ad-dl309 production in any of these cell lines, 2′AP strongly increased production of both Ad-dl1520 and AdΔE1b in parental HepG2 cells as well as HepG2-E1b-Mut cells. As expected, HepG2-E1b-WT at least partially complemented the E1b deletions in Ad-dl1520 and AdΔE1b allowing relatively high virus production, and 2′AP treatment did not further increase yields substantially. Interestingly, AdΔE1b production levels were much lower than Ad-dl1520 even in the complementing HepG2-E1b-WT cells, suggesting that other differences between Ad-dl1520 and AdΔE1b, such as E1b-19K and the E3 death protein encoded by Ad-dl1520, or mCMV-promoter-control of E1a in AdΔE1b, may also contributed to lower AdΔE1b production.

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