Rad51 Targeted Adenoviral Vector Figure 7. Expression of a toxic gene from the Rad51core promoter causes little toxicity in primary cells. Primary cells (as indicated) were transduced with high doses of a replication-defective Ad5 vector encoding the HSV TK gene under the transcriptional control of the Rad51core element (Ad5DE1-Rad51core-TK) or the constitutively active CMV promoter (Ad5DE1-CMV-TK). Cells were transduced with the vector at the indicated MOIs (5–500), in the presence or absence of varying concentrations of ganciclovir (0–200 mM). Five days later, cell viability was determined with AlamarBlueTM dye and normalized as a percentage of the mean value of untreated cells. Data are presented as mean values of independent experimental triplicates; error bars represent the standard deviation of the data. doi:10.1371/journal.pone.0028714.g007 activated Rad51 promoter activity. The fact that p300 overex- pression increased Rad51core promoter activity even in the absence of p53 further supports the idea that p300 is limiting with respect to the transcriptional activity of the Rad51core promoter, and suggests that some additional inhibitory factors may be acting on this promoter in a p300-dependent manner. It has been previously suggested that competition by transcrip- tion factors for binding to limiting amounts of p300/CBP protein may lead to transcriptional repression of one of the competing gene targets [32,33]. Furthermore p53 has been shown to transcriptionally repress target genes through interaction with p300/CBP binding partners, such as Ets1/2 [34], Sp1 [35] and C/EBP [36]. Analysis of the Rad51core promoter using the Transcription Element Search System (TESS) [37], revealed potential binding sites for the transcription factors Sp1, C/EBP, and E2F-1. Hasselbach et al. also reported binding sites and DNA binding by E2F-1 as well as by STAT-5 within the Rad51 promoter [22], and Rad51 expression has been shown to be regulated by E2F-1 [38] and STAT5 [39]. Since all of these transcription factors can be activated through interactions with p300/CBP [25,40], competitive binding of p53 to p300/CBP offers a likely mechanism through which p53 may repress the Rad51 core promoter. Alternatively Rad51 may be also regulated by the many changes to cellular metabolism that occur during oncogenic transformation, some of which may be the result of a p53 defect. Finally, we demonstrated the ability of a Rad51core driven adenoviral vector to achieve selective cell killing of p53-defective cancer cells (Figure 6), while sparing normal cells (Figure 7). The Ad5DE1-Rad51core-TK and Ad5DE1-CMV-TK vectors were PLoS ONE | www.plosone.org 9 December 2011 | Volume 6 | Issue 12 | e28714