Limits...
Inhibition of histone deacetylation and DNA methylation improves gene expression mediated by the adeno-associated virus/phage in cancer cells.

Kia A, Yata T, Hajji N, Hajitou A - Viruses (2013)

Bottom Line: We previously designed an improved phage vector by incorporating cis genetic elements of adeno-associated virus (AAV).Using a combination of AAVP expressing the GFP reporter gene, flow cytometry, inhibitors of histone deacetylation, and DNA methylation, we have demonstrated that histone deacetylation and DNA methylation are associated with silencing of gene expression from the CMV promoter in the parental AAVP.However, cell confluence had no effect on AAVP-guided gene expression.

View Article: PubMed Central - PubMed

Affiliation: Phage Therapy Group, Department of Medicine, Imperial College London, Hammersmith Hospital Campus, London W12 0NN, UK. a.hajitou@imperial.ac.uk.

ABSTRACT
Bacteriophage (phage), viruses that infect bacteria only, have become promising vectors for targeted systemic delivery of genes to cancer, although, with poor efficiency. We previously designed an improved phage vector by incorporating cis genetic elements of adeno-associated virus (AAV). This novel AAV/phage hybrid (AAVP) specifically targeted systemic delivery of therapeutic genes into tumors. To advance the AAVP vector, we recently introduced the stress-inducible Grp78 tumor specific promoter and found that this dual tumor-targeted AAVP provides persistent gene expression, over time, in cancer cells compared to silenced gene expression from the CMV promoter in the parental AAVP. Herein, we investigated the effect of histone deacetylation and DNA methylation on AAVP-mediated gene expression in cancer cells and explored the effect of cell confluence state on AAVP gene expression efficacy. Using a combination of AAVP expressing the GFP reporter gene, flow cytometry, inhibitors of histone deacetylation, and DNA methylation, we have demonstrated that histone deacetylation and DNA methylation are associated with silencing of gene expression from the CMV promoter in the parental AAVP. Importantly, inhibitors of histone deacetylases boost gene expression in cancer cells from the Grp78 promoter in the dual tumor-targeted AAVP. However, cell confluence had no effect on AAVP-guided gene expression. Our findings prove that combination of histone deacetylase inhibitor drugs with the Grp78 promoter is an effective approach to improve AAVP-mediated gene expression in cancer cells and should be considered for AAVP-based clinical cancer gene therapy.

Show MeSH

Related in: MedlinePlus

Persistence of gene expression from RGD4C/AAVP-Grp78 and silencing of RGD4C/AAVP-CMV-mediated gene delivery in cancer cells. U87 and 9L glioblastoma cells were stably transduced with RGD4C/AAVP-CMV-GFP or RGD4C/AAVP-Grp78-GFP vectors. Then GFP positive cells were monitored by flow cytometry over a period of 39 to 75 days post-transduction of U87 cells, and 39 to 97 days post-transduction of 9L cells. This experiment was repeated three times with similar results, shown are data of one experiment. Statistical analyses were performed by using GraphPad Prism software (version 5.0). Error bars represent standard error of the mean (s.e.m). p-values were generated by ANOVA and denoted as follows: *p < 0.05, **p < 0.01 and ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3814604&req=5

viruses-05-02561-f001: Persistence of gene expression from RGD4C/AAVP-Grp78 and silencing of RGD4C/AAVP-CMV-mediated gene delivery in cancer cells. U87 and 9L glioblastoma cells were stably transduced with RGD4C/AAVP-CMV-GFP or RGD4C/AAVP-Grp78-GFP vectors. Then GFP positive cells were monitored by flow cytometry over a period of 39 to 75 days post-transduction of U87 cells, and 39 to 97 days post-transduction of 9L cells. This experiment was repeated three times with similar results, shown are data of one experiment. Statistical analyses were performed by using GraphPad Prism software (version 5.0). Error bars represent standard error of the mean (s.e.m). p-values were generated by ANOVA and denoted as follows: *p < 0.05, **p < 0.01 and ***p < 0.001.

Mentions: We monitored gene expression by AAVP in the human U87 and rat 9L glioblastoma cells over an extended time course by generating stably transduced cells with vectors carrying puroR gene that confers puromycin resistance. A marked decrease in gene expression from the RGD4C/AAVP-CMV phage vector was observed over time in U87 and 9L cells; in contrast, no silencing of Grp78-regulated gene expression was detected following cell transduction with the double-targeted RGD4C/AAVP-Grp78 phage (Figure 1).


Inhibition of histone deacetylation and DNA methylation improves gene expression mediated by the adeno-associated virus/phage in cancer cells.

Kia A, Yata T, Hajji N, Hajitou A - Viruses (2013)

Persistence of gene expression from RGD4C/AAVP-Grp78 and silencing of RGD4C/AAVP-CMV-mediated gene delivery in cancer cells. U87 and 9L glioblastoma cells were stably transduced with RGD4C/AAVP-CMV-GFP or RGD4C/AAVP-Grp78-GFP vectors. Then GFP positive cells were monitored by flow cytometry over a period of 39 to 75 days post-transduction of U87 cells, and 39 to 97 days post-transduction of 9L cells. This experiment was repeated three times with similar results, shown are data of one experiment. Statistical analyses were performed by using GraphPad Prism software (version 5.0). Error bars represent standard error of the mean (s.e.m). p-values were generated by ANOVA and denoted as follows: *p < 0.05, **p < 0.01 and ***p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814604&req=5

viruses-05-02561-f001: Persistence of gene expression from RGD4C/AAVP-Grp78 and silencing of RGD4C/AAVP-CMV-mediated gene delivery in cancer cells. U87 and 9L glioblastoma cells were stably transduced with RGD4C/AAVP-CMV-GFP or RGD4C/AAVP-Grp78-GFP vectors. Then GFP positive cells were monitored by flow cytometry over a period of 39 to 75 days post-transduction of U87 cells, and 39 to 97 days post-transduction of 9L cells. This experiment was repeated three times with similar results, shown are data of one experiment. Statistical analyses were performed by using GraphPad Prism software (version 5.0). Error bars represent standard error of the mean (s.e.m). p-values were generated by ANOVA and denoted as follows: *p < 0.05, **p < 0.01 and ***p < 0.001.
Mentions: We monitored gene expression by AAVP in the human U87 and rat 9L glioblastoma cells over an extended time course by generating stably transduced cells with vectors carrying puroR gene that confers puromycin resistance. A marked decrease in gene expression from the RGD4C/AAVP-CMV phage vector was observed over time in U87 and 9L cells; in contrast, no silencing of Grp78-regulated gene expression was detected following cell transduction with the double-targeted RGD4C/AAVP-Grp78 phage (Figure 1).

Bottom Line: We previously designed an improved phage vector by incorporating cis genetic elements of adeno-associated virus (AAV).Using a combination of AAVP expressing the GFP reporter gene, flow cytometry, inhibitors of histone deacetylation, and DNA methylation, we have demonstrated that histone deacetylation and DNA methylation are associated with silencing of gene expression from the CMV promoter in the parental AAVP.However, cell confluence had no effect on AAVP-guided gene expression.

View Article: PubMed Central - PubMed

Affiliation: Phage Therapy Group, Department of Medicine, Imperial College London, Hammersmith Hospital Campus, London W12 0NN, UK. a.hajitou@imperial.ac.uk.

ABSTRACT
Bacteriophage (phage), viruses that infect bacteria only, have become promising vectors for targeted systemic delivery of genes to cancer, although, with poor efficiency. We previously designed an improved phage vector by incorporating cis genetic elements of adeno-associated virus (AAV). This novel AAV/phage hybrid (AAVP) specifically targeted systemic delivery of therapeutic genes into tumors. To advance the AAVP vector, we recently introduced the stress-inducible Grp78 tumor specific promoter and found that this dual tumor-targeted AAVP provides persistent gene expression, over time, in cancer cells compared to silenced gene expression from the CMV promoter in the parental AAVP. Herein, we investigated the effect of histone deacetylation and DNA methylation on AAVP-mediated gene expression in cancer cells and explored the effect of cell confluence state on AAVP gene expression efficacy. Using a combination of AAVP expressing the GFP reporter gene, flow cytometry, inhibitors of histone deacetylation, and DNA methylation, we have demonstrated that histone deacetylation and DNA methylation are associated with silencing of gene expression from the CMV promoter in the parental AAVP. Importantly, inhibitors of histone deacetylases boost gene expression in cancer cells from the Grp78 promoter in the dual tumor-targeted AAVP. However, cell confluence had no effect on AAVP-guided gene expression. Our findings prove that combination of histone deacetylase inhibitor drugs with the Grp78 promoter is an effective approach to improve AAVP-mediated gene expression in cancer cells and should be considered for AAVP-based clinical cancer gene therapy.

Show MeSH
Related in: MedlinePlus