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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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Schematic representation of the adenoviral genomes used in this study.Ad-dl309 encodes both E1a and E1b genes including their respective Ad promoters (pE1a and pE1b, respectively). The virus has a deletion in the E3 region spanning 30005–30750 bp [81]. Ad-dl309ΔVA and Ad-dl1520 have additional deletions in the VA-RNA genes (10667–10702 bp as well as 10929–10943 bp) or in the E1b-55K gene, respectively. AdΔE1b, AdΔE1bΔVA and AdControl (ΔE1aΔE1b) were constructed using the AdEasy-1 system and the pAdTrack shuttle plasmid. The pAd-Easy-1 plasmid has a larger deletion in the E3 region spanning 28130–30820 bp. The pAd-Track plasmid has E1 sequences spanning 480–3533 bp replaced with the EGFP gene under the control of the human cytomegalovirus immediate early (hCMV) promoter [53]. The E1a gene under the control of the murine cytomegalovirus immediate early (mCMV) promoter was introduced back into the genomes of AdΔE1b and AdΔE1bΔVA. Additionally, AdΔE1bΔVA has the same VA-RNA deletions as Ad-dl309ΔVA.
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pone-0065222-g001: Schematic representation of the adenoviral genomes used in this study.Ad-dl309 encodes both E1a and E1b genes including their respective Ad promoters (pE1a and pE1b, respectively). The virus has a deletion in the E3 region spanning 30005–30750 bp [81]. Ad-dl309ΔVA and Ad-dl1520 have additional deletions in the VA-RNA genes (10667–10702 bp as well as 10929–10943 bp) or in the E1b-55K gene, respectively. AdΔE1b, AdΔE1bΔVA and AdControl (ΔE1aΔE1b) were constructed using the AdEasy-1 system and the pAdTrack shuttle plasmid. The pAd-Easy-1 plasmid has a larger deletion in the E3 region spanning 28130–30820 bp. The pAd-Track plasmid has E1 sequences spanning 480–3533 bp replaced with the EGFP gene under the control of the human cytomegalovirus immediate early (hCMV) promoter [53]. The E1a gene under the control of the murine cytomegalovirus immediate early (mCMV) promoter was introduced back into the genomes of AdΔE1b and AdΔE1bΔVA. Additionally, AdΔE1bΔVA has the same VA-RNA deletions as Ad-dl309ΔVA.

Mentions: Ad-dl309, Ad-dl309ΔVA (dVAs) and the non-replicating AdControl (rAd-gal-GFP) [27], [52] were kind gifts from Dr. Matthias Dobbelstein (Göttingen University). Ad-dl1520 [1] was a kind gift from Dr. Philip Branton (McGill University). AdΔE1b and AdΔE1bΔVA were constructed using the AdEasy-1 system [53]. Briefly, Ad5 E1a gene (excluding viral E1a promoter) was PCR amplified from the plasmid pXC1 (nucleotides 542–1564; a gift from Dr. Frank Graham). This fragment was added to an expression vector encoding a PacI-deleted murine cytomegalovirus immediate early (mCMV) promoter. The expression cassette was then transferred into the pAd-Track shuttle vector. This pAd-Track plasmid was then recombined in BJ5183 bacterial cells with the pAd-Easy-1 plasmid or a derivative lacking the VA-RNA genes [27]. The resulting recombinant plasmids were cleaved with PacI to release the viral genome and used to transfect HEK293. Recombinant viruses were isolated and amplified in Hep3B cells due to the reduced growth of VA-RNA deleted viruses in HEK293 cells. Ad-dl309 and Ad-dl309ΔVA were also amplified in Hep3B cells, while the replication-deficient AdControl was amplified in HEK293 cells. All viruses (Figure 1) were purified by CsCl gradient sedimentation [54] and their genomes were isolated and verified by restriction enzyme digestion. The concentration of virus particles was determined spectrophotometrically [54]. The concentration of infectious virus was determined through plaque assays performed on HEK293 cells as previously described [54]. Due to the inability of AdΔE1bΔVA to produce plaques in HEK293 cells, the concentration of infectious units of AdΔE1bΔVA as well as AdΔE1b were determined from the number of GFP-positive Hep3B cells observed 3 days after infection with limiting dilutions of virus (designated green fluorescent units, GFU). The ratios of virus particle to infectious units of AdΔE1b and AdΔE1bΔVA were approximately 70, similar to the particle to PFU ratio for the other viruses.


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)

Schematic representation of the adenoviral genomes used in this study.Ad-dl309 encodes both E1a and E1b genes including their respective Ad promoters (pE1a and pE1b, respectively). The virus has a deletion in the E3 region spanning 30005–30750 bp [81]. Ad-dl309ΔVA and Ad-dl1520 have additional deletions in the VA-RNA genes (10667–10702 bp as well as 10929–10943 bp) or in the E1b-55K gene, respectively. AdΔE1b, AdΔE1bΔVA and AdControl (ΔE1aΔE1b) were constructed using the AdEasy-1 system and the pAdTrack shuttle plasmid. The pAd-Easy-1 plasmid has a larger deletion in the E3 region spanning 28130–30820 bp. The pAd-Track plasmid has E1 sequences spanning 480–3533 bp replaced with the EGFP gene under the control of the human cytomegalovirus immediate early (hCMV) promoter [53]. The E1a gene under the control of the murine cytomegalovirus immediate early (mCMV) promoter was introduced back into the genomes of AdΔE1b and AdΔE1bΔVA. Additionally, AdΔE1bΔVA has the same VA-RNA deletions as Ad-dl309ΔVA.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672087&req=5

pone-0065222-g001: Schematic representation of the adenoviral genomes used in this study.Ad-dl309 encodes both E1a and E1b genes including their respective Ad promoters (pE1a and pE1b, respectively). The virus has a deletion in the E3 region spanning 30005–30750 bp [81]. Ad-dl309ΔVA and Ad-dl1520 have additional deletions in the VA-RNA genes (10667–10702 bp as well as 10929–10943 bp) or in the E1b-55K gene, respectively. AdΔE1b, AdΔE1bΔVA and AdControl (ΔE1aΔE1b) were constructed using the AdEasy-1 system and the pAdTrack shuttle plasmid. The pAd-Easy-1 plasmid has a larger deletion in the E3 region spanning 28130–30820 bp. The pAd-Track plasmid has E1 sequences spanning 480–3533 bp replaced with the EGFP gene under the control of the human cytomegalovirus immediate early (hCMV) promoter [53]. The E1a gene under the control of the murine cytomegalovirus immediate early (mCMV) promoter was introduced back into the genomes of AdΔE1b and AdΔE1bΔVA. Additionally, AdΔE1bΔVA has the same VA-RNA deletions as Ad-dl309ΔVA.
Mentions: Ad-dl309, Ad-dl309ΔVA (dVAs) and the non-replicating AdControl (rAd-gal-GFP) [27], [52] were kind gifts from Dr. Matthias Dobbelstein (Göttingen University). Ad-dl1520 [1] was a kind gift from Dr. Philip Branton (McGill University). AdΔE1b and AdΔE1bΔVA were constructed using the AdEasy-1 system [53]. Briefly, Ad5 E1a gene (excluding viral E1a promoter) was PCR amplified from the plasmid pXC1 (nucleotides 542–1564; a gift from Dr. Frank Graham). This fragment was added to an expression vector encoding a PacI-deleted murine cytomegalovirus immediate early (mCMV) promoter. The expression cassette was then transferred into the pAd-Track shuttle vector. This pAd-Track plasmid was then recombined in BJ5183 bacterial cells with the pAd-Easy-1 plasmid or a derivative lacking the VA-RNA genes [27]. The resulting recombinant plasmids were cleaved with PacI to release the viral genome and used to transfect HEK293. Recombinant viruses were isolated and amplified in Hep3B cells due to the reduced growth of VA-RNA deleted viruses in HEK293 cells. Ad-dl309 and Ad-dl309ΔVA were also amplified in Hep3B cells, while the replication-deficient AdControl was amplified in HEK293 cells. All viruses (Figure 1) were purified by CsCl gradient sedimentation [54] and their genomes were isolated and verified by restriction enzyme digestion. The concentration of virus particles was determined spectrophotometrically [54]. The concentration of infectious virus was determined through plaque assays performed on HEK293 cells as previously described [54]. Due to the inability of AdΔE1bΔVA to produce plaques in HEK293 cells, the concentration of infectious units of AdΔE1bΔVA as well as AdΔE1b were determined from the number of GFP-positive Hep3B cells observed 3 days after infection with limiting dilutions of virus (designated green fluorescent units, GFU). The ratios of virus particle to infectious units of AdΔE1b and AdΔE1bΔVA were approximately 70, similar to the particle to PFU ratio for the other viruses.

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