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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.

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Related in: MedlinePlus

HDAC inhibitors restore gene expression from RGD4C/AAVP-CMV and boost RGD4C/AAVP-Grp78-mediated gene expression in cancer cells. U87 cells at day 75 post-transduction with (A) RGD4C/AAVP-CMV-GFP or (B) RGD4C/AAVP-Grp78-GFP were plated in 6 well plates, then treated 24 h later with various concentrations of the HDAC inhibitors TSA, SAHA, nicotinamide or VPA for three days; (C) 9L cells stably transduced with RGD4C/AAVP-Grp78-GFP were treated with various concentrations of the HDAC inhibitors at day 97 post-transduction as described above. Finally, U87 and 9L cells were collected and GFP positive cells as well as the MFI were analyzed by flow cytometry. The experiments were performed in triplicate, repeated three times with similar results, and a representative experiment is shown. 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.
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viruses-05-02561-f002: HDAC inhibitors restore gene expression from RGD4C/AAVP-CMV and boost RGD4C/AAVP-Grp78-mediated gene expression in cancer cells. U87 cells at day 75 post-transduction with (A) RGD4C/AAVP-CMV-GFP or (B) RGD4C/AAVP-Grp78-GFP were plated in 6 well plates, then treated 24 h later with various concentrations of the HDAC inhibitors TSA, SAHA, nicotinamide or VPA for three days; (C) 9L cells stably transduced with RGD4C/AAVP-Grp78-GFP were treated with various concentrations of the HDAC inhibitors at day 97 post-transduction as described above. Finally, U87 and 9L cells were collected and GFP positive cells as well as the MFI were analyzed by flow cytometry. The experiments were performed in triplicate, repeated three times with similar results, and a representative experiment is shown. 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: Although the exact mechanisms of viral promoter silencing have remained mainly unknown, several studies have demonstrated the association of DNA methylation and histone deacetylation with inactivation of the CMV promoter [11,13,16,17]. Generally, both DNA methylation and histone acetylation statuses play major roles in the regulation of gene expression by providing transcription factors’ accessibility to gene promoters. The precise balance of acetylated and deacetylated states of histones is an important feature of gene regulation and the imbalance is found in many human cancers, often resulting from alterations in histone acetyltransferase (HATs) and histone deacetylase (HDACs) enzyme activities. Here, we quantified AAVP-mediated gene expression in the presence of HDAC inhibitors by using vectors expressing the green fluorescent protein (GFP), RGD4C/AAVP-CMV-GFP and RGD4C/AAVP-Grp78-GFP, and carrying the puroR to generate stable gene expression by stably transduced cells. Flow cytometry was used and both percentage of GFP positive cells and mean fluorescent intensity (MFI) were calculated by normalizing the results to parental non-transduced cells. As an initial experiment, we evaluated GFP expression in the human U87 cancer cells transduced with RGD4C/AAVP-CMV-GFP or RGD4C/AAVP-Grp78-GFP upon treatment with increasing concentrations of trichostatin-A (TSA), a pan-HDAC inhibitor. TSA is the first characterized organic HDAC inhibitor [18] widely utilized to study the reactivation of silenced viral constructs. In RGD4C/AAVP-CMV-GFP-transduced U87 cells, treatment with 0.5 µM and 1 µM TSA resulted in a significant increase of both percentage of GFP positive cells by 1.4- and 1.8-fold, and MFI by 2.7- and 3.0-fold, respectively (Figure 2A). These findings are consistent with previous reports demonstrating reactivation of the CMV promoter by TSA in U87 cells and other cell lines [12,19]. Interestingly, GFP expression in U87 cells stably transduced by RGD4C/AAVP-Grp78-GFP increased at the level of MFI only, upon TSA treatment, with no effect on GFP positive cells (Figure 2B). Next, we investigated additional HDAC inhibitors such as suberoylanilide hydroxamic acid (SAHA), which is structurally similar to TSA, as well as nicotinamide and valporic acid (VPA). SAHA treatment with 0.5 µM and 1 µM yielded results comparable to TSA and resulted in a dose dependent reactivation of gene expression in U87 cells transduced with RGD4C/AAVP-CMV-GFP, enhancing the percentage of GFP positive cells by 1.6- and 2.2-fold, as well as MFI by 1.8- and 2.8-fold, respectively (Figure 2A). Interestingly, in RGD4C/AAVP-Grp78-GFP-transduced cells and similar to TSA treatment, only the MFI was increased upon SAHA treatment while the percentage of GFP positive cells remained intact (Figure 2B). Finally, treatment with either nicotinamide or VPA had no effect on GFP expression of U87 cells transduced with either RGD4C/AAVP-CMV-GFP (Figure 2A) or RGD4C/AAVP-Grp78-GFP (Figure 2B). These results show that TSA and SAHA, both Zn2+ binding inhibitors of HDACs class I and II, restore GFP expression from RGD4C/AAVP-CMV in U87 cells; whereas, nicotinamide, a class III HDAC inhibitor, and VPA, an inhibitor of class I HDACs, had no effect on gene expression from the RGD4C/AAVP-CMV-GFP phage vector. Based on these observations we can postulate the involvement of HDAC class II in gene expression silencing from RGD4C/AAVP-CMV-GFP in U87 cells. Interestingly, induction of RGD4C/AAVP-Grp78-guided gene expression by TSA and SAHA treatment was also reproduced in the 9L cells transduced with RGD4C/AAVP-Grp78-GFP, and consistently with U87 cells, only MFI was increased with no effect on GFP positive cells (Figure 2C). Again, nicotinamide and VPA inhibitors had no impact (Figure 2C). In contrast, TSA, SAHA, nicotinamide, and VPA treatment of 9L cells transduced with RGD4C/AAVP-CMV-GFP had no significant effect on GFP expression (data not shown). Consequently, we investigated an additional mechanism regulating the CMV promoter silencing, the promoter methylation status, to determine whether gene expression from the RGD4C/AAVP-CMV phage in 9L cells can be rescued by DNA methylation inhibitors. Thus, treatment with various concentrations of the DNA methylation inhibitor 5-Azacytidine (5-Aza) resulted in a dose dependent increase of GFP expression in 9L cells transduced with RGD4C/AAVP-CMV-GFP (Figure 3A). Addition of 20 µM of 5-Aza increased the percentage of GFP positive cells to 17.5% compared to 3.5% of control untreated cells, and boosted the MFI by 8.9-fold. In contrast, 5-Aza treatment of 9L cells transduced with the RGD4C/AAVP-Grp78-GFP phage resulted in no significant change in GFP expression (Figure 3B). Extensive methylation of the CMV promoter sequences has previously been reported [12,13,16,20]. Chromatin structural alterations in the CMV promoter region were also described following suppression of expression of genes stably delivered by AAV vectors carrying the CMV promoter [21,22].


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)

HDAC inhibitors restore gene expression from RGD4C/AAVP-CMV and boost RGD4C/AAVP-Grp78-mediated gene expression in cancer cells. U87 cells at day 75 post-transduction with (A) RGD4C/AAVP-CMV-GFP or (B) RGD4C/AAVP-Grp78-GFP were plated in 6 well plates, then treated 24 h later with various concentrations of the HDAC inhibitors TSA, SAHA, nicotinamide or VPA for three days; (C) 9L cells stably transduced with RGD4C/AAVP-Grp78-GFP were treated with various concentrations of the HDAC inhibitors at day 97 post-transduction as described above. Finally, U87 and 9L cells were collected and GFP positive cells as well as the MFI were analyzed by flow cytometry. The experiments were performed in triplicate, repeated three times with similar results, and a representative experiment is shown. 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-f002: HDAC inhibitors restore gene expression from RGD4C/AAVP-CMV and boost RGD4C/AAVP-Grp78-mediated gene expression in cancer cells. U87 cells at day 75 post-transduction with (A) RGD4C/AAVP-CMV-GFP or (B) RGD4C/AAVP-Grp78-GFP were plated in 6 well plates, then treated 24 h later with various concentrations of the HDAC inhibitors TSA, SAHA, nicotinamide or VPA for three days; (C) 9L cells stably transduced with RGD4C/AAVP-Grp78-GFP were treated with various concentrations of the HDAC inhibitors at day 97 post-transduction as described above. Finally, U87 and 9L cells were collected and GFP positive cells as well as the MFI were analyzed by flow cytometry. The experiments were performed in triplicate, repeated three times with similar results, and a representative experiment is shown. 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: Although the exact mechanisms of viral promoter silencing have remained mainly unknown, several studies have demonstrated the association of DNA methylation and histone deacetylation with inactivation of the CMV promoter [11,13,16,17]. Generally, both DNA methylation and histone acetylation statuses play major roles in the regulation of gene expression by providing transcription factors’ accessibility to gene promoters. The precise balance of acetylated and deacetylated states of histones is an important feature of gene regulation and the imbalance is found in many human cancers, often resulting from alterations in histone acetyltransferase (HATs) and histone deacetylase (HDACs) enzyme activities. Here, we quantified AAVP-mediated gene expression in the presence of HDAC inhibitors by using vectors expressing the green fluorescent protein (GFP), RGD4C/AAVP-CMV-GFP and RGD4C/AAVP-Grp78-GFP, and carrying the puroR to generate stable gene expression by stably transduced cells. Flow cytometry was used and both percentage of GFP positive cells and mean fluorescent intensity (MFI) were calculated by normalizing the results to parental non-transduced cells. As an initial experiment, we evaluated GFP expression in the human U87 cancer cells transduced with RGD4C/AAVP-CMV-GFP or RGD4C/AAVP-Grp78-GFP upon treatment with increasing concentrations of trichostatin-A (TSA), a pan-HDAC inhibitor. TSA is the first characterized organic HDAC inhibitor [18] widely utilized to study the reactivation of silenced viral constructs. In RGD4C/AAVP-CMV-GFP-transduced U87 cells, treatment with 0.5 µM and 1 µM TSA resulted in a significant increase of both percentage of GFP positive cells by 1.4- and 1.8-fold, and MFI by 2.7- and 3.0-fold, respectively (Figure 2A). These findings are consistent with previous reports demonstrating reactivation of the CMV promoter by TSA in U87 cells and other cell lines [12,19]. Interestingly, GFP expression in U87 cells stably transduced by RGD4C/AAVP-Grp78-GFP increased at the level of MFI only, upon TSA treatment, with no effect on GFP positive cells (Figure 2B). Next, we investigated additional HDAC inhibitors such as suberoylanilide hydroxamic acid (SAHA), which is structurally similar to TSA, as well as nicotinamide and valporic acid (VPA). SAHA treatment with 0.5 µM and 1 µM yielded results comparable to TSA and resulted in a dose dependent reactivation of gene expression in U87 cells transduced with RGD4C/AAVP-CMV-GFP, enhancing the percentage of GFP positive cells by 1.6- and 2.2-fold, as well as MFI by 1.8- and 2.8-fold, respectively (Figure 2A). Interestingly, in RGD4C/AAVP-Grp78-GFP-transduced cells and similar to TSA treatment, only the MFI was increased upon SAHA treatment while the percentage of GFP positive cells remained intact (Figure 2B). Finally, treatment with either nicotinamide or VPA had no effect on GFP expression of U87 cells transduced with either RGD4C/AAVP-CMV-GFP (Figure 2A) or RGD4C/AAVP-Grp78-GFP (Figure 2B). These results show that TSA and SAHA, both Zn2+ binding inhibitors of HDACs class I and II, restore GFP expression from RGD4C/AAVP-CMV in U87 cells; whereas, nicotinamide, a class III HDAC inhibitor, and VPA, an inhibitor of class I HDACs, had no effect on gene expression from the RGD4C/AAVP-CMV-GFP phage vector. Based on these observations we can postulate the involvement of HDAC class II in gene expression silencing from RGD4C/AAVP-CMV-GFP in U87 cells. Interestingly, induction of RGD4C/AAVP-Grp78-guided gene expression by TSA and SAHA treatment was also reproduced in the 9L cells transduced with RGD4C/AAVP-Grp78-GFP, and consistently with U87 cells, only MFI was increased with no effect on GFP positive cells (Figure 2C). Again, nicotinamide and VPA inhibitors had no impact (Figure 2C). In contrast, TSA, SAHA, nicotinamide, and VPA treatment of 9L cells transduced with RGD4C/AAVP-CMV-GFP had no significant effect on GFP expression (data not shown). Consequently, we investigated an additional mechanism regulating the CMV promoter silencing, the promoter methylation status, to determine whether gene expression from the RGD4C/AAVP-CMV phage in 9L cells can be rescued by DNA methylation inhibitors. Thus, treatment with various concentrations of the DNA methylation inhibitor 5-Azacytidine (5-Aza) resulted in a dose dependent increase of GFP expression in 9L cells transduced with RGD4C/AAVP-CMV-GFP (Figure 3A). Addition of 20 µM of 5-Aza increased the percentage of GFP positive cells to 17.5% compared to 3.5% of control untreated cells, and boosted the MFI by 8.9-fold. In contrast, 5-Aza treatment of 9L cells transduced with the RGD4C/AAVP-Grp78-GFP phage resulted in no significant change in GFP expression (Figure 3B). Extensive methylation of the CMV promoter sequences has previously been reported [12,13,16,20]. Chromatin structural alterations in the CMV promoter region were also described following suppression of expression of genes stably delivered by AAV vectors carrying the CMV promoter [21,22].

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