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The HDAC inhibitor FK228 enhances adenoviral transgene expression by a transduction-independent mechanism but does not increase adenovirus replication.

Danielsson A, Dzojic H, Rashkova V, Cheng WS, Essand M - PLoS ONE (2011)

Bottom Line: In the present study, we found that the highest increase in transgene expression was achieved when non-toxic concentrations of FK228 were added immediately after transduction, demonstrating that the main effect by which FK228 enhances transgene expression is transduction-independent.In some cases, the effects were dramatic, as demonstrated by an increase in CD40L expression by FK228 from 0.3% to 62% when the murine prostate cancer cell line TRAMP-C2 was transduced with Ad[CD40L].One unexpected finding was that FK228 decreased the transgene expression of an adenoviral vector with the prostate cell-specific PPT promoter in the human prostate adenocarcinoma cell lines LNCaP and PC-346C.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden. angelika.danielsson@igp.uu.se

ABSTRACT
The histone deacetylase inhibitor FK228 has previously been shown to enhance adenoviral transgene expression when cells are pre-incubated with the drug. Upregulation of the coxsackie adenovirus receptor (CAR), leading to increased viral transduction, has been proposed as the main mechanism. In the present study, we found that the highest increase in transgene expression was achieved when non-toxic concentrations of FK228 were added immediately after transduction, demonstrating that the main effect by which FK228 enhances transgene expression is transduction-independent. FK228 had positive effects both on Ad5 and Ad5/f35 vectors with a variety of transgenes and promoters, indicating that FK228 works mainly by increasing transgene expression at the transcriptional level. In some cases, the effects were dramatic, as demonstrated by an increase in CD40L expression by FK228 from 0.3% to 62% when the murine prostate cancer cell line TRAMP-C2 was transduced with Ad[CD40L]. One unexpected finding was that FK228 decreased the transgene expression of an adenoviral vector with the prostate cell-specific PPT promoter in the human prostate adenocarcinoma cell lines LNCaP and PC-346C. This is probably a consequence of alteration of the adenocarcinoma cell lines towards a neuroendocrine differentiation after FK228 treatment. The observations in this study indicate that FK228 enhances adenoviral therapy by a transduction-independent mechanism. Furthermore, since histone deacetylase inhibitors may affect the differentiation of cells, it is important to keep in mind that the activity and specificity of tissue- and tumor-specific promoters may also be affected.

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HDAC inhibitors affect tissue-specific promoters differently.A) BON cells were transduced with Ad[CgA-LUC] and Ad[CMV-LUC] and LNCaP and PC-346C were transduced with Ad[i/PPT-LUC], Ad[CgA-LUC] and Ad[CMV-LUC]. Cells were then incubated with 3 ng/ml FK228 or 5 mM VPA for 48 h. Both FK228 and VPA enhanced the transgene expression of the Ad[CgA-LUC] vector in BON cells about 10 times although the increase was not significant. In LNCaP and PC-346C, the expression of Ad[i/PPT-LUC] was significantly decreased while the expression of Ad[CgA-LUC] was significantly increased by HDACi treatment. B) LNCaP and PC-346C cells were transduced with Ad[I/PPT-GFP] and Ad[CMV-GFP] and then incubated with 3 ng/ml FK228 or 5 mM VPA for 48 h followed by flow cytometry analysis. The percentages of GFP positive cells (upper right) and MFI values (lower right) are given. GFP levels were decreased by HDAC inhibitors when the transgene expression was controlled by the PPT promoter. One representative experiment out of three is shown. Significance (p<0.05) is indicated with asterisks.
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pone-0014700-g005: HDAC inhibitors affect tissue-specific promoters differently.A) BON cells were transduced with Ad[CgA-LUC] and Ad[CMV-LUC] and LNCaP and PC-346C were transduced with Ad[i/PPT-LUC], Ad[CgA-LUC] and Ad[CMV-LUC]. Cells were then incubated with 3 ng/ml FK228 or 5 mM VPA for 48 h. Both FK228 and VPA enhanced the transgene expression of the Ad[CgA-LUC] vector in BON cells about 10 times although the increase was not significant. In LNCaP and PC-346C, the expression of Ad[i/PPT-LUC] was significantly decreased while the expression of Ad[CgA-LUC] was significantly increased by HDACi treatment. B) LNCaP and PC-346C cells were transduced with Ad[I/PPT-GFP] and Ad[CMV-GFP] and then incubated with 3 ng/ml FK228 or 5 mM VPA for 48 h followed by flow cytometry analysis. The percentages of GFP positive cells (upper right) and MFI values (lower right) are given. GFP levels were decreased by HDAC inhibitors when the transgene expression was controlled by the PPT promoter. One representative experiment out of three is shown. Significance (p<0.05) is indicated with asterisks.

Mentions: After finding out that FK228 increases transgene expression from various viral vectors, we wanted to investigate if FK228 had the same positive effect on adenoviral vectors with tissue-specific promoters as it has on an adenoviral vectors with the CMV promoter. The human chromogranin A (CgA) promoter was positively affected, although not significantly, by FK228 and VPA when the neuroendocrine pancreatic carcinoid cell line BON was transduced with Ad[CgA-LUC] (Figure 5A). A vector with the same transgene driven by the CMV promoter was used for comparison. Next, LNCaP and PC-346C cells were transduced with Ad[i/PPT-LUC and Ad[i/PPT-GFP] and thereafter incubated with FK228 or VPA for 48 h. Interestingly, the PPT-driven transgene expression of both luciferase (Figure 5A) and GFP (Figure 5B) was decreased when the cells were incubated with either of the two HDACi while, as before, CMV-driven GFP expression was increased when cells were treated with FK228 after transduction. VPA treatment increased the expression of both luciferase and GFP when the CMV promoter was used (Figure 5A and 5B). Of further interest, when the prostate cancer cell lines LNCaP and PC346-C were transduced with the neuroendocrine-selective Ad[CgA-LUC] vector and then treated with FK228 or VPA, we found that transgene expression was increased.


The HDAC inhibitor FK228 enhances adenoviral transgene expression by a transduction-independent mechanism but does not increase adenovirus replication.

Danielsson A, Dzojic H, Rashkova V, Cheng WS, Essand M - PLoS ONE (2011)

HDAC inhibitors affect tissue-specific promoters differently.A) BON cells were transduced with Ad[CgA-LUC] and Ad[CMV-LUC] and LNCaP and PC-346C were transduced with Ad[i/PPT-LUC], Ad[CgA-LUC] and Ad[CMV-LUC]. Cells were then incubated with 3 ng/ml FK228 or 5 mM VPA for 48 h. Both FK228 and VPA enhanced the transgene expression of the Ad[CgA-LUC] vector in BON cells about 10 times although the increase was not significant. In LNCaP and PC-346C, the expression of Ad[i/PPT-LUC] was significantly decreased while the expression of Ad[CgA-LUC] was significantly increased by HDACi treatment. B) LNCaP and PC-346C cells were transduced with Ad[I/PPT-GFP] and Ad[CMV-GFP] and then incubated with 3 ng/ml FK228 or 5 mM VPA for 48 h followed by flow cytometry analysis. The percentages of GFP positive cells (upper right) and MFI values (lower right) are given. GFP levels were decreased by HDAC inhibitors when the transgene expression was controlled by the PPT promoter. One representative experiment out of three is shown. Significance (p<0.05) is indicated with asterisks.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040751&req=5

pone-0014700-g005: HDAC inhibitors affect tissue-specific promoters differently.A) BON cells were transduced with Ad[CgA-LUC] and Ad[CMV-LUC] and LNCaP and PC-346C were transduced with Ad[i/PPT-LUC], Ad[CgA-LUC] and Ad[CMV-LUC]. Cells were then incubated with 3 ng/ml FK228 or 5 mM VPA for 48 h. Both FK228 and VPA enhanced the transgene expression of the Ad[CgA-LUC] vector in BON cells about 10 times although the increase was not significant. In LNCaP and PC-346C, the expression of Ad[i/PPT-LUC] was significantly decreased while the expression of Ad[CgA-LUC] was significantly increased by HDACi treatment. B) LNCaP and PC-346C cells were transduced with Ad[I/PPT-GFP] and Ad[CMV-GFP] and then incubated with 3 ng/ml FK228 or 5 mM VPA for 48 h followed by flow cytometry analysis. The percentages of GFP positive cells (upper right) and MFI values (lower right) are given. GFP levels were decreased by HDAC inhibitors when the transgene expression was controlled by the PPT promoter. One representative experiment out of three is shown. Significance (p<0.05) is indicated with asterisks.
Mentions: After finding out that FK228 increases transgene expression from various viral vectors, we wanted to investigate if FK228 had the same positive effect on adenoviral vectors with tissue-specific promoters as it has on an adenoviral vectors with the CMV promoter. The human chromogranin A (CgA) promoter was positively affected, although not significantly, by FK228 and VPA when the neuroendocrine pancreatic carcinoid cell line BON was transduced with Ad[CgA-LUC] (Figure 5A). A vector with the same transgene driven by the CMV promoter was used for comparison. Next, LNCaP and PC-346C cells were transduced with Ad[i/PPT-LUC and Ad[i/PPT-GFP] and thereafter incubated with FK228 or VPA for 48 h. Interestingly, the PPT-driven transgene expression of both luciferase (Figure 5A) and GFP (Figure 5B) was decreased when the cells were incubated with either of the two HDACi while, as before, CMV-driven GFP expression was increased when cells were treated with FK228 after transduction. VPA treatment increased the expression of both luciferase and GFP when the CMV promoter was used (Figure 5A and 5B). Of further interest, when the prostate cancer cell lines LNCaP and PC346-C were transduced with the neuroendocrine-selective Ad[CgA-LUC] vector and then treated with FK228 or VPA, we found that transgene expression was increased.

Bottom Line: In the present study, we found that the highest increase in transgene expression was achieved when non-toxic concentrations of FK228 were added immediately after transduction, demonstrating that the main effect by which FK228 enhances transgene expression is transduction-independent.In some cases, the effects were dramatic, as demonstrated by an increase in CD40L expression by FK228 from 0.3% to 62% when the murine prostate cancer cell line TRAMP-C2 was transduced with Ad[CD40L].One unexpected finding was that FK228 decreased the transgene expression of an adenoviral vector with the prostate cell-specific PPT promoter in the human prostate adenocarcinoma cell lines LNCaP and PC-346C.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden. angelika.danielsson@igp.uu.se

ABSTRACT
The histone deacetylase inhibitor FK228 has previously been shown to enhance adenoviral transgene expression when cells are pre-incubated with the drug. Upregulation of the coxsackie adenovirus receptor (CAR), leading to increased viral transduction, has been proposed as the main mechanism. In the present study, we found that the highest increase in transgene expression was achieved when non-toxic concentrations of FK228 were added immediately after transduction, demonstrating that the main effect by which FK228 enhances transgene expression is transduction-independent. FK228 had positive effects both on Ad5 and Ad5/f35 vectors with a variety of transgenes and promoters, indicating that FK228 works mainly by increasing transgene expression at the transcriptional level. In some cases, the effects were dramatic, as demonstrated by an increase in CD40L expression by FK228 from 0.3% to 62% when the murine prostate cancer cell line TRAMP-C2 was transduced with Ad[CD40L]. One unexpected finding was that FK228 decreased the transgene expression of an adenoviral vector with the prostate cell-specific PPT promoter in the human prostate adenocarcinoma cell lines LNCaP and PC-346C. This is probably a consequence of alteration of the adenocarcinoma cell lines towards a neuroendocrine differentiation after FK228 treatment. The observations in this study indicate that FK228 enhances adenoviral therapy by a transduction-independent mechanism. Furthermore, since histone deacetylase inhibitors may affect the differentiation of cells, it is important to keep in mind that the activity and specificity of tissue- and tumor-specific promoters may also be affected.

Show MeSH
Related in: MedlinePlus