Effective Transgene Constructs to Enhance Gene Therapy with Trichostatin A

Hayashi, Hideki; Ma, Yue; Kohno, Tomoko; Igarashi, Masayuki; Yasui, Kiyoshi; Chua, Yoshinao Kubo Koon Jiew; Ishibashi, Masayoshi; Urae, Ryuji; Irie, Shin; Matsuyama, Tomohiro

doi:10.5772/18523

Cited by 2 publications

(2 citation statements)

References 53 publications

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“…Since the luciferase and EGFP plasmids used in this study rely on different promoters (SV40 and CMV, respectively), these results suggest that Entinostat may be able to enhance a variety of different therapeutic genes and promoters. These results agree with previous findings that the pan‐HDAC (HDAC 1, 3, 4, 6, and 10) inhibitor Trichostatin A significantly enhances expression from a variety of viral promoters, including the SV40 and CMV promoters used in this study (Barua and Rege, ; Hayashi et al, ). The highest average luciferase expression values were obtained when Entinostat was used with PEI, but the other polymers tested (1,4C‐1,4Bis and PA8) also showed significant degrees of enhancement.…”

Section: Discussionsupporting

confidence: 93%

The histone deacetylase inhibitor Entinostat enhances polymer‐mediated transgene expression in cancer cell lines

Elmer

Christensen

Barua

et al. 2015

Biotech & Bioengineering

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Eukaryotic cells maintain an immense amount of genetic information by tightly wrapping their DNA around positively charged histones. While this strategy allows human cells to maintain more than 25,000 genes, histone binding can also block gene expression. Consequently, cells express histone acetyl transferases (HATs) to acetylate histone lysines and release DNA for transcription. Conversely, histone deacetylases (HDACs) are employed for restoring the positive charge on the histones, thereby silencing gene expression by increasing histone-DNA binding. It has previously been shown that histones bind and silence viral DNA, while hyperacetylation of histones via HDAC inhibition restores viral gene expression. In this study, we demonstrate that treatment with Entinostat, an HDAC inhibitor, enhances transgene (luciferase) expression by up to 25-fold in human prostate and murine bladder cancer cell lines when used with cationic polymers for plasmid DNA delivery. Entinostat treatment altered cell cycle progression, resulting in a significant increase in the fraction of cells present in the G0/G1 phase at low micromolar concentrations. While this moderate G0/G1 arrest disappeared at higher concentrations, a modest increase in the fraction of apoptotic cells and a decrease in cell proliferation were observed, consistent with the known anticancer effects of the drug. DNase accessibility studies revealed no significant change in plasmid transcriptional availability with Entinostat treatment. However, quantitative PCR studies indicated that Entinostat treatment, at the optimal dose for enhancing transgene expression, led to an increase in the amount of plasmid present in the nucleus in two cancer cell lines. Taken together, our results show that Entinostat enhances polymer-mediated transgene expression and can be useful in applications related to transient protein expression in mammalian cells.

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Section: Discussionsupporting

confidence: 93%

The histone deacetylase inhibitor Entinostat enhances polymer‐mediated transgene expression in cancer cell lines

Elmer

Christensen

Barua

et al. 2015

Biotech & Bioengineering

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“…However, since TSA (Trichostatin A) activated the PGK promoter and showed a concentration-dependent increase in Luc2 activity, we decided to use the cell viability data for standardization. In fact, it has been reported that TSA activates the promoters of housekeeping genes such as CMV (Hayashi et al, 2011), so we thought that data standardization using the promoters of housekeeping genes was not ideal. Therefore, in order to obtain cell viability data, we had to prepare separate cell plates treated under the same conditions, which resulted in increased experimental effort.…”

Section: Improving Points For Future Usementioning

confidence: 99%

Development of a new <i>in vitro</i> assay system for evaluating the effects of chemicals on DNA methylation

et al. 2021

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Epigenetic toxicity, a phenomenon in which chemicals exert epigenetic effects and produce toxicity, has been attracting attention in recent years due to advances in toxicology accompanying the development of life sciences. However, it has been difficult to identify epigenetic toxicants due to the lack of a simple experimental system to evaluate epigenetic toxicity. In this study, we developed a prototype of an in vitro reporter assay system for assessing the effects of chemicals on DNA methylation using two promoters showing different degrees of DNA methylation, Agouti IAP and Daz1 promoters, and a luciferase reporter. The system successfully detected DNA demethylating activity using 5-azacytidine, a chemical having DNA demethylation activity, as a positive control chemical, and demethylation of cytosine of CpG in the promoter was confirmed by pyrosequencing analysis. Next, in order to improve the detection sensitivity of the DNA demethylating activity of this system, we tried to increase the basal level of methylation of the Daz1 promoter by pre-methylase treatment of the reporter vectors. As a result, the detection sensitivity of the system was successfully improved in cells where the basal level of methylation was indeed increased by methylase treatment. Thus, the developed assay system here is effective for the simple evaluation of chemicals that affect DNA methylation.

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Effective Transgene Constructs to Enhance Gene Therapy with Trichostatin A

Cited by 2 publications

References 53 publications

The histone deacetylase inhibitor Entinostat enhances polymer‐mediated transgene expression in cancer cell lines

The histone deacetylase inhibitor Entinostat enhances polymer‐mediated transgene expression in cancer cell lines

Development of a new <i>in vitro</i> assay system for evaluating the effects of chemicals on DNA methylation

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