Rad51 Targeted Adenoviral Vector Figure 6. The Rad51core promoter can be used to selectively express a toxic gene in p53-defective cells. P53 defective cancer cells (as indicated) were transduced with a replication-defective Ad5 vector encoding the HSV thymidine kinase (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 or 50), in the presence or absence of varying concentrations of ganciclovir (0–100 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.g006 the tumor suppressor p53, one of the most commonly mutated genes in cancer [27]. Our data show that p53 suppresses the activity of the Rad51 promoter, and that Rad51 promoter activity in different cell lines has an inverse relationship to p53 function. Two exceptions to this trend were noted: A549 cells (where the Rad51 promoter was highly active) and BxPc-3 cells (where activity was lower than in other p53-defective cells). We tentatively attribute this to additional regulatory dysfunction in these cancer lines. A549 cells have been reported to have an overexpression of MDM-2 [28], which promotes nuclear export [29] and degrada- tion of p53 protein [30]. As a consequence, Lu et al. were unable to detect p53 in A549 cells by immunocytochemical staining, and they reported low levels of p53 in the cell even after transduction with exogenous p53 DNA. This suggests that, while the p53 gene is normal in A549 cells, protein expression levels are extremely low – and possibly too low to effectively suppress Rad51-driven gene expression. In the case of the BxPc-3 pancreatic cancer cell line, we confirmed that the previously described mutation at codon 220 of the p53 gene in this cell line was indeed present [31]. However, BxPc-3 cells have been shown to express high levels of mutant p53 [3,31]. Thus, one possible explanation for our data is that the overabundance of this mutant isoform of p53 in BxPc-3 cells actually serves to further sequester p300 (resulting in low levels of Rad51-driven luciferase expression). Additionally, pancreatic cancer cells have been shown to accurately reflect their malignant phenotype [3], in which Rad51 is overexpressed, only when cultured in a three dimensional matrix. Overall, our results are broadly consistent with a previous report that p53 can suppress the transcriptional activity of the Rad51 promoter [24]. Contrary to this study, however, we found that p53-mediated suppression of Rad51 promoter activity occurred independently of the reported p53 binding region in the promoter [24]. This may reflect methodological differences, since we studied the core Rad51 promoter (2230/+217) in the context of a linear adenoviral vector, whereas Arias-Lopez et al. studied a larger fragment of the promoter (2948/+1427) in the context of a circular plasmid vector [24]. Regardless, our data strongly suggest that p53 can act an indirect manner, possibly through effects on other transcription factors or cofactors that may regulate the Rad51 promoter. p300 is a well known co-activator of transcription, shown to interact with p53 and many other transcription factors [25]. Here we showed that overexpression of p300 increased transcriptional activity from the Rad51core promoter and relieved the p53 mediated repression (Figure 5). This suggests that p53 may have sequestered limiting amounts of p300 that otherwise could have PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e28714