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Truncating the i-leader open reading frame enhances release of human adenovirus type 5 in glioma cells.

van den Hengel SK, de Vrij J, Uil TG, Lamfers ML, Sillevis Smitt PA, Hoeben RC - Virol. J. (2011)

Bottom Line: The survival of glioma patients with the current treatments is poor.A mutation truncating the i-leader open reading frame was created in a molecular clone of replication-competent wild-type HAdV-5 by site-directed mutagenesis.Such mutations may help enhancing the antitumor cytopathic efficacy of oncolytic adenoviruses in the treatment of glioblastoma.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, P.O. Box 9600, 2300 RC, Leiden, The Netherlands. r.c.hoeben@lumc.nl.

ABSTRACT

Background: The survival of glioma patients with the current treatments is poor. Early clinical trails with replicating adenoviruses demonstrated the feasibility and safety of the use of adenoviruses as oncolytic agents. Antitumor efficacy has been moderate due to inefficient virus replication and spread. Previous studies have shown that truncation of the adenovirus i-leader open reading frame enhanced cytopathic activity of HAdV-5 in several tumor cell lines. Here we report the effect of an i-leader mutation on the cytopathic activity in glioma cell lines and in primary high-grade glioma cell cultures.

Results: A mutation truncating the i-leader open reading frame was created in a molecular clone of replication-competent wild-type HAdV-5 by site-directed mutagenesis. We analyzed the cytopathic activity of this RL-07 mutant virus. A cell-viability assay showed increased cytopathic activity of the RL-07 mutant virus on U251 and SNB19 glioma cell lines. The plaque sizes of RL-07 on U251 monolayers were seven times larger than those of isogenic control viruses. Similarly, the cytopathic activity of the RL-07 viruses was strongly increased in six primary high-grade glioma cell cultures. In glioma cell lines the RL-07 virus was found to be released earlier into the culture medium. This was not due to enhanced viral protein synthesis, as was evident from equivalent E1A, Fiber and Adenovirus Death Protein amounts, nor to higher virus yields.

Conclusion: The cytopathic activity of replicating adenovirus in glioblastoma cells is increased by truncating the i-leader open reading frame. Such mutations may help enhancing the antitumor cytopathic efficacy of oncolytic adenoviruses in the treatment of glioblastoma.

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Related in: MedlinePlus

Protein immunoblot assay for detection of viral proteins. Fiber, E1A, ADP levels were detected upon infection with RL-07 or wtHAdV-5 at various time points p.i.. Actin was used as loading control. Cells were seeded o/n in a 6-well plate and infected with MOI = 1. Cells were harvested 24, 40, 48, 64 and 70 hrs p.i. in Ripa buffer. Protein amounts were determined by BCA protein assay. In each lane 50 μg of protein was loaded. C-mock infected control; W- infected with wtHAdV-5; R- infected with RL-07.
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Figure 4: Protein immunoblot assay for detection of viral proteins. Fiber, E1A, ADP levels were detected upon infection with RL-07 or wtHAdV-5 at various time points p.i.. Actin was used as loading control. Cells were seeded o/n in a 6-well plate and infected with MOI = 1. Cells were harvested 24, 40, 48, 64 and 70 hrs p.i. in Ripa buffer. Protein amounts were determined by BCA protein assay. In each lane 50 μg of protein was loaded. C-mock infected control; W- infected with wtHAdV-5; R- infected with RL-07.

Mentions: Having established that the RL-07 virus was released earlier than wtHAdV-5, we examined the expression pattern of the viral proteins E1A, fiber, and the adenovirus death protein (ADP). U251 and SNB19 cells were infected at MOI = 1 and harvested at several time points after infection and viral proteins were visualized by immunoblotting (Figure 4). In our time series no differences between fiber, E1A and ADP levels were observed between RL-07 and wtHAdV-5. This shows that the rapid release of RL-07 particles does not result from more rapid viral protein synthesis.


Truncating the i-leader open reading frame enhances release of human adenovirus type 5 in glioma cells.

van den Hengel SK, de Vrij J, Uil TG, Lamfers ML, Sillevis Smitt PA, Hoeben RC - Virol. J. (2011)

Protein immunoblot assay for detection of viral proteins. Fiber, E1A, ADP levels were detected upon infection with RL-07 or wtHAdV-5 at various time points p.i.. Actin was used as loading control. Cells were seeded o/n in a 6-well plate and infected with MOI = 1. Cells were harvested 24, 40, 48, 64 and 70 hrs p.i. in Ripa buffer. Protein amounts were determined by BCA protein assay. In each lane 50 μg of protein was loaded. C-mock infected control; W- infected with wtHAdV-5; R- infected with RL-07.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3090740&req=5

Figure 4: Protein immunoblot assay for detection of viral proteins. Fiber, E1A, ADP levels were detected upon infection with RL-07 or wtHAdV-5 at various time points p.i.. Actin was used as loading control. Cells were seeded o/n in a 6-well plate and infected with MOI = 1. Cells were harvested 24, 40, 48, 64 and 70 hrs p.i. in Ripa buffer. Protein amounts were determined by BCA protein assay. In each lane 50 μg of protein was loaded. C-mock infected control; W- infected with wtHAdV-5; R- infected with RL-07.
Mentions: Having established that the RL-07 virus was released earlier than wtHAdV-5, we examined the expression pattern of the viral proteins E1A, fiber, and the adenovirus death protein (ADP). U251 and SNB19 cells were infected at MOI = 1 and harvested at several time points after infection and viral proteins were visualized by immunoblotting (Figure 4). In our time series no differences between fiber, E1A and ADP levels were observed between RL-07 and wtHAdV-5. This shows that the rapid release of RL-07 particles does not result from more rapid viral protein synthesis.

Bottom Line: The survival of glioma patients with the current treatments is poor.A mutation truncating the i-leader open reading frame was created in a molecular clone of replication-competent wild-type HAdV-5 by site-directed mutagenesis.Such mutations may help enhancing the antitumor cytopathic efficacy of oncolytic adenoviruses in the treatment of glioblastoma.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, P.O. Box 9600, 2300 RC, Leiden, The Netherlands. r.c.hoeben@lumc.nl.

ABSTRACT

Background: The survival of glioma patients with the current treatments is poor. Early clinical trails with replicating adenoviruses demonstrated the feasibility and safety of the use of adenoviruses as oncolytic agents. Antitumor efficacy has been moderate due to inefficient virus replication and spread. Previous studies have shown that truncation of the adenovirus i-leader open reading frame enhanced cytopathic activity of HAdV-5 in several tumor cell lines. Here we report the effect of an i-leader mutation on the cytopathic activity in glioma cell lines and in primary high-grade glioma cell cultures.

Results: A mutation truncating the i-leader open reading frame was created in a molecular clone of replication-competent wild-type HAdV-5 by site-directed mutagenesis. We analyzed the cytopathic activity of this RL-07 mutant virus. A cell-viability assay showed increased cytopathic activity of the RL-07 mutant virus on U251 and SNB19 glioma cell lines. The plaque sizes of RL-07 on U251 monolayers were seven times larger than those of isogenic control viruses. Similarly, the cytopathic activity of the RL-07 viruses was strongly increased in six primary high-grade glioma cell cultures. In glioma cell lines the RL-07 virus was found to be released earlier into the culture medium. This was not due to enhanced viral protein synthesis, as was evident from equivalent E1A, Fiber and Adenovirus Death Protein amounts, nor to higher virus yields.

Conclusion: The cytopathic activity of replicating adenovirus in glioblastoma cells is increased by truncating the i-leader open reading frame. Such mutations may help enhancing the antitumor cytopathic efficacy of oncolytic adenoviruses in the treatment of glioblastoma.

Show MeSH
Related in: MedlinePlus