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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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Relative survival as determined by WST-1 assay. Cytopathic activity of RL-07 (grey bar) compared to wtHAdV-5 (black bar) is higher on glioma cell lines U251 and SNB19. Error bars represent the SD (n = 3). * - statistical significance; unpaired, two-sided t-test, p < 0.01. Cells were seeded in 96-well plates 1 day before infection with RL-07 and wtHAd5 (MOI range 0-100 pfu/cell). Viability of the cultures was determined 4 days p.i by WST-1 assay. Very similar results were obtained in a repeat experiment.
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Figure 1: Relative survival as determined by WST-1 assay. Cytopathic activity of RL-07 (grey bar) compared to wtHAdV-5 (black bar) is higher on glioma cell lines U251 and SNB19. Error bars represent the SD (n = 3). * - statistical significance; unpaired, two-sided t-test, p < 0.01. Cells were seeded in 96-well plates 1 day before infection with RL-07 and wtHAd5 (MOI range 0-100 pfu/cell). Viability of the cultures was determined 4 days p.i by WST-1 assay. Very similar results were obtained in a repeat experiment.

Mentions: To analyze the cytopathic activity of RL-07, two glioma cell lines (U251 and SNB19, both CAR-positive) and two control cell lines (911 and A549) were infected with RL-07 and wtHAdV-5 at multiplicity of infections (MOI) ranging from 0 to 100 pfu/cell. Four days p.i., the cell viability was determined by WST-1 assay (Figure 1), RL-07 had a significant higher cytopathic effect on glioma cell lines and A549 compared to the control virus wtHAdV-5. On 911 cells, no differences in the induction of cytopathic effects were detected.


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)

Relative survival as determined by WST-1 assay. Cytopathic activity of RL-07 (grey bar) compared to wtHAdV-5 (black bar) is higher on glioma cell lines U251 and SNB19. Error bars represent the SD (n = 3). * - statistical significance; unpaired, two-sided t-test, p < 0.01. Cells were seeded in 96-well plates 1 day before infection with RL-07 and wtHAd5 (MOI range 0-100 pfu/cell). Viability of the cultures was determined 4 days p.i by WST-1 assay. Very similar results were obtained in a repeat experiment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Relative survival as determined by WST-1 assay. Cytopathic activity of RL-07 (grey bar) compared to wtHAdV-5 (black bar) is higher on glioma cell lines U251 and SNB19. Error bars represent the SD (n = 3). * - statistical significance; unpaired, two-sided t-test, p < 0.01. Cells were seeded in 96-well plates 1 day before infection with RL-07 and wtHAd5 (MOI range 0-100 pfu/cell). Viability of the cultures was determined 4 days p.i by WST-1 assay. Very similar results were obtained in a repeat experiment.
Mentions: To analyze the cytopathic activity of RL-07, two glioma cell lines (U251 and SNB19, both CAR-positive) and two control cell lines (911 and A549) were infected with RL-07 and wtHAdV-5 at multiplicity of infections (MOI) ranging from 0 to 100 pfu/cell. Four days p.i., the cell viability was determined by WST-1 assay (Figure 1), RL-07 had a significant higher cytopathic effect on glioma cell lines and A549 compared to the control virus wtHAdV-5. On 911 cells, no differences in the induction of cytopathic effects were detected.

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