The HDAC Inhibitor FK228 Enhances Adenoviral Transgene Expression by a Transduction-Independent Mechanism but Does Not Increase Adenovirus Replication Angelika Danielsson*, Helena Dzojic, Victoria Rashkova, Wing-Shing Cheng, Magnus Essand Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden 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. Citation: Danielsson A, Dzojic H, Rashkova V, Cheng W-S, Essand M (2011) The HDAC Inhibitor FK228 Enhances Adenoviral Transgene Expression by a Transduction-Independent Mechanism but Does Not Increase Adenovirus Replication. PLoS ONE 6(2): e14700. doi:10.1371/journal.pone.0014700 Editor: Venugopalan Cheriyath, Cleveland Clinic, United States of America Received July 23, 2010; Accepted January 25, 2011; Published February 17, 2011 Copyright: 2011 Danielsson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by funding from The Swedish Cancer Society (Grant CAN 2007/885 and CAN 2009/55), the Swedish Research Council (Grant K2008-68X-15270-04-3), Gunnar Nilsson’s Cancer Foundation (Grant 65/09) and the European Community on behalf of GIANT (Grant LSHB-CT-2004-512087). ME is a recipient of the Swedish Cancer Society Senior Investigator Award. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: angelika.danielsson@igp.uu.se Introduction Viral vectors based on human adenovirus serotype 5 (Ad5) are the most commonly used vectors for gene therapy and they are frequently used for vaccination. In cancer gene therapy their effect can be limited due to that malignant cells often have low expression of the coxsackie adenovirus receptor (CAR), which is the primary receptor for Ad5 [1]. Different means to increase CAR expression on target cells would presumably lead to enhanced adenoviral gene therapy efficacy. Regulation of gene expression is controlled by various mechanisms. Histone acetylation plays a key role in transcriptional control by modulating chromatin structure [2]. In general, histone acetylation is associated with relaxation of chromatin and activation of transcription whereas histone deacetylation is associated with condensation of the chromatin structure and repression of transcription. Histone deacetylase inhibitors (HDACi) cause accumulation of acetylated histones, which affect transcription of specific genes and both up- and down-regulation of gene expression can occur [3]. FK228, also known as depsipeptide or romidepsin, is an HDACi that can induce cell cycle arrest, promote apoptosis and inhibit angiogenesis [4]. FK228 triggers both mitochondrial-dependent [5] and mitochon- drial-independent [6] apoptosis and is more toxic to malignant than non-malignant cells [7]. Because of its direct cytotoxic effects, FK228 has been and is currently being evaluated in clinical cancer trials [8,9]. FK228 has minimal antitumor activity in patients with metastatic castration-resistant prostate cancer [10]. In combina- tion with docetaxel however, FK228 is able to enhance the antitumor effect both in vitro and in prostate cancer xenograft mouse models [11,12]. In addition, FK228 has been shown to enhance the effect of adenoviral-mediated therapy [13,14]. The mechanisms for this enhancement are not fully understood but upregulation of CAR has been suggested as one possibility [7,15,16,17,18]. Herein, we investigate the effect that FK228 has on adenoviral transduction and transgene expression in cancer cells. Upregula- tion of CAR plays if any, only a minimal role in the enhancement of transgene expression. Instead, we show that FK228 has the highest effect when administered directly after transduction, PLoS ONE | www.plosone.org 1 February 2011 | Volume 6 | Issue 2 | e14700