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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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FK228 enhances transgene expression in various cell types.Cells were transduced with Ad[CMV-GFP] or Ad5/f35[CMV-GFP] for 2 h and then incubated for 48 h in medium containing 3 ng/ml FK228. The percentages of GFP positive cells (upper right) are given as well as MFI values (lower right or left). FK228 enhanced transgene expression of Ad5 virus and Ad5 virus with fiber shaft and knob from serotype 35 in all cell lines. Each cell line was evaluated three times with similar results. Monocytes from five different donors were evaluated. Representative examples are displayed in the figure.
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pone-0014700-g003: FK228 enhances transgene expression in various cell types.Cells were transduced with Ad[CMV-GFP] or Ad5/f35[CMV-GFP] for 2 h and then incubated for 48 h in medium containing 3 ng/ml FK228. The percentages of GFP positive cells (upper right) are given as well as MFI values (lower right or left). FK228 enhanced transgene expression of Ad5 virus and Ad5 virus with fiber shaft and knob from serotype 35 in all cell lines. Each cell line was evaluated three times with similar results. Monocytes from five different donors were evaluated. Representative examples are displayed in the figure.

Mentions: We next wanted to investigate the effect of FK228 on adenoviral transduction. LNCaP, PC-346C and TRAMP-C2 were grown in medium with or without 3 ng/ml FK228 for 24 h. Subsequently, cells were transduced with Ad[CMV-GFP] for 2 h and then cultured in medium with or without FK228 for another 48 h. GFP expression was analyzed by flow cytometry. Pre-incubation of cells with FK228 enhanced GFP expression to some extent in LNCaP and TRAMP-C2 (Figure 2). Pre- and post-incubation gave an even higher level of GFP expression in all three cell lines. However, the highest increase was observed when FK228 was added after transduction, indicating a post-transductional effect of FK228. The fact that FK228 had the largest effect when given after transduction, both when looking at percentage of GFP positive cells and mean fluorescence intensity (MFI), indicates that the receptors involved in adenoviral transduction may play a less important role for enhanced transgene expression as previously proposed. Next, we used both the serotype 5-based vector Ad[CMV-GFP] and the adenoviral serotype 5 vector with fiber shaft and knob from serotype 35 Ad5/f35[CMV-GFP] to screen cell lines of various origin as well as freshly isolated human monocytes. Treatment of cells with FK228 after transduction enhanced GFP expression (both percentage and MFI) in all cell lines and monocytes for both viruses (Figure 3). The effect was not as pronounced for a lentiviral vector with regard to GFP positive cells as FK228 increased GFP expression of LN[CMV-GFP] in TRAMP-C2 cells but had no effect in the other cell lines tested (Figure S1). The MFI values on the other hand were increased in all cells except for BON and monocytes.


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)

FK228 enhances transgene expression in various cell types.Cells were transduced with Ad[CMV-GFP] or Ad5/f35[CMV-GFP] for 2 h and then incubated for 48 h in medium containing 3 ng/ml FK228. The percentages of GFP positive cells (upper right) are given as well as MFI values (lower right or left). FK228 enhanced transgene expression of Ad5 virus and Ad5 virus with fiber shaft and knob from serotype 35 in all cell lines. Each cell line was evaluated three times with similar results. Monocytes from five different donors were evaluated. Representative examples are displayed in the figure.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040751&req=5

pone-0014700-g003: FK228 enhances transgene expression in various cell types.Cells were transduced with Ad[CMV-GFP] or Ad5/f35[CMV-GFP] for 2 h and then incubated for 48 h in medium containing 3 ng/ml FK228. The percentages of GFP positive cells (upper right) are given as well as MFI values (lower right or left). FK228 enhanced transgene expression of Ad5 virus and Ad5 virus with fiber shaft and knob from serotype 35 in all cell lines. Each cell line was evaluated three times with similar results. Monocytes from five different donors were evaluated. Representative examples are displayed in the figure.
Mentions: We next wanted to investigate the effect of FK228 on adenoviral transduction. LNCaP, PC-346C and TRAMP-C2 were grown in medium with or without 3 ng/ml FK228 for 24 h. Subsequently, cells were transduced with Ad[CMV-GFP] for 2 h and then cultured in medium with or without FK228 for another 48 h. GFP expression was analyzed by flow cytometry. Pre-incubation of cells with FK228 enhanced GFP expression to some extent in LNCaP and TRAMP-C2 (Figure 2). Pre- and post-incubation gave an even higher level of GFP expression in all three cell lines. However, the highest increase was observed when FK228 was added after transduction, indicating a post-transductional effect of FK228. The fact that FK228 had the largest effect when given after transduction, both when looking at percentage of GFP positive cells and mean fluorescence intensity (MFI), indicates that the receptors involved in adenoviral transduction may play a less important role for enhanced transgene expression as previously proposed. Next, we used both the serotype 5-based vector Ad[CMV-GFP] and the adenoviral serotype 5 vector with fiber shaft and knob from serotype 35 Ad5/f35[CMV-GFP] to screen cell lines of various origin as well as freshly isolated human monocytes. Treatment of cells with FK228 after transduction enhanced GFP expression (both percentage and MFI) in all cell lines and monocytes for both viruses (Figure 3). The effect was not as pronounced for a lentiviral vector with regard to GFP positive cells as FK228 increased GFP expression of LN[CMV-GFP] in TRAMP-C2 cells but had no effect in the other cell lines tested (Figure S1). The MFI values on the other hand were increased in all cells except for BON and monocytes.

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