Adenoviral Vector Driven by a Minimal Rad51 Promoter Is Selective for p53-Deficient Tumor Cells Vincent Fong 1 , Marika Osterbur1, Cristina Capella1, Yo-El Kim1, Christopher Hine2, Vera Gorbunova2, Andrei Seluanov2, Stephen Dewhurst1* 1 Department of Microbiology and Immunology, University of Rochester, Rochester, New York, United States of America, 2 Department of Biology, University of Rochester, Rochester, New York, United States of America Abstract Background: The full length Rad51 promoter is highly active in cancer cells but not in normal cells. We therefore set out to assess whether we could confer this tumor-selectivity to an adenovirus vector. Methodology/Principal Findings: Expression of an adenovirally-vectored luciferase reporter gene from the Rad51 promoter was up to 50 fold higher in cancer cells than in normal cells. Further evaluations of a panel of truncated promoter mutants identified a 447 bp minimal core promoter element that retained the full tumor selectivity and transcriptional activity of the original promoter, in the context of an adenovirus vector. This core Rad51 promoter was highly active in cancer cells that lack functional p53, but less active in normal cells and in cancer cell lines with intact p53 function. Exogenous expression of p53 in a p53 cell line strongly suppressed activity of the Rad51 core promoter, underscoring the selectivity of this promoter for p53-deficient cells. Follow-up experiments showed that the p53-dependent suppression of the Rad51 core promoter was mediated via an indirect, p300 coactivator dependent mechanism. Finally, transduction of target cells with an adenovirus vector encoding the thymidine kinase gene under transcriptional control of the Rad51 core promoter resulted in efficient killing of p53 defective cancer cells, but not of normal cells, upon addition of ganciclovir. Conclusions/Significance: Overall, these experiments demonstrated that a small core domain of the Rad51 promoter can be used to target selective transgene expression from adenoviral vectors to tumor cells lacking functional p53. Citation: Fong V, Osterbur M, Capella C, Kim Y-E, Hine C, et al. (2011) Adenoviral Vector Driven by a Minimal Rad51 Promoter Is Selective for p53-Deficient Tumor Cells. PLoS ONE 6(12): e28714. doi:10.1371/journal.pone.0028714 Editor: Volker Thiel, Kantonal Hospital St. Gallen, Switzerland Received June 14, 2011; Accepted November 14, 2011; Published December 9, 2011 Copyright: 2011 Fong 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: VF is a trainee in the Medical Scientist Training Program, funded by the U.S. National Institutes of Health (NIH) award T32 GM07356. This work was also supported by NIH awards R21 CA122213 (SD), R01 AG031227, R01 AG27237 (VG) and T32 GM068411 (VF). The funders had no role in 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: stephen_dewhurst@urmc.rochester.edu Introduction Specific targeting of therapeutic agents to cancer cells while avoiding damage to normal tissue has been a long time goal in cancer research. One method of targeting viral agents has been to use tumor specific promoters to restrict expression of therapeutic genes [1,2]. Expression of the DNA repair gene, Rad51, has been shown to be upregulated in many cancers [3,4,5], especially higher grade [6,7,8,9] chemoresistant [10] and radioresistant tumors [11]. The Rad51 protein plays a key role in homologous recombination [12]. Expression is tightly regulated in normal cells, with dysregulation leading to genomic instability and possibly contributing to oncogenesis [13,14,15,16,17]. Recently, Gorbunova and colleagues reported that the full length Rad51 promoter maintains its cancer specificity when taken independent of its natural context and showed that it can drive tumor-selective expression of a reporter gene [18]. This makes the Rad51 promoter a very attractive candidate for use in anti-cancer therapies especially when coupled with the efficient transduction capabilities of viral vectors [19]. We therefore conducted experiments to examine the feasibility of using the Rad51 promoter to drive tumor-selective expression of a transgene of interest from an adenovirus vector. An essential initial objective was to define the minimal Rad51 promoter element that retained the robust transcriptional activity and tumor selectivity of the intact promoter, since the full length Rad51 promoter reported by Gorbunova and colleagues is over 6.5 kb in length [18] and exceeds the insert capacity for many adenoviral vectors [20,21]. Our experiments succeeded in identifying a minimal core promoter element of approximately 450 bp that retained the full tumor selectivity and transcriptional activity of the intact promoter. We also found that the Rad51 promoter was more active in cancer cells that lacked functional p53, compared to cells with normal p53 (including both normal cells and cancer cells with intact p53 function). We then proceeded to evaluate the ability of this minimal core promoter to drive selective expression of the herpes simplex virus type 1 (HSV) thymidine kinase (TK) gene from an adenoviral vector in p53 defective cancer cells. Our studies showed ganciclovir dependent killing of transduced p53 defective cells with little effect on normal cells. These data suggest that the Rad51 core promoter may have utility in virally vectored gene therapies for p53 defective cancers. PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e28714