Rad51 Targeted Adenoviral Vector Known p53 defect Description Citation Table 1. Description of cell lines used in this study. Cell Lines Normal cell lines Cancer cell lines Wi-38 HCA-2 BJ MRC-5 SAEC nHeps A549 BxPc-3 MCF-7 H1299 HeLa SF-539 U251 None None None None None None None None p53null None Mutation Y220C Likely degradation by HPV E6 Mutation R273H Normal lung fibroblasts Foreskin fibroblasts immortalized with hTERT Foreskin fibroblasts Normal lung fibroblasts Normal Small Airway Epithelial Cells (Clonetics) Normal human primary hepatocytes (Clonetics) Lung carcinoma Pancreatic adenocarcinoma Breast adenocarcinoma Non-small cell lung carcinoma Cervical adenocarcinoma Glioma Glioma (Primary cell) (Primary cell) [42] [43] [45] [47] [46] [31] [46] [48] [44] [46] [46] doi:10.1371/journal.pone.0028714.t001 function. To formally test this hypothesis, we segregated our panel of 9 cell lines into lines previously reported to have defective or deleted p53, and those reported to have normal p53 function (Table 1). The median luciferase activity in cells transduced with Ad5DE1-Rad51full-luc was 272,000 RLU/20 mg cell lysate for cells with wild-type p53. In contrast, median promoter activity in cells lacking functional p53 was 10-fold higher, at 2,790,000 RLU/20 mg cell lysate (Figure 4A). Using a Mann-Whitney analysis we found that Rad51 promoter activity differed significantly between the 2 groups (p-value = 0.03; Figure 4A). In order to confirm the inverse relationship between Rad51 promoter activity and p53 function, we transfected a p53-null cell line, H1299, with a plasmid encoding a p53-GFP fusion protein and then 12 hrs later transduced the same cells with Ad5DE1- Rad51core-luc or Ad5DE1-CMV-luc. After a further 24 hours, the cells were then flow cytometrically sorted into GFP+ and GFP2 populations, lysed and luciferase activity was measured. When p53 function was restored to H1299 cells, Rad51core promoter activity was reduced to less than 15% of its original activity, while there was no significant change in CMV promoter activity (Figure 4B). This suggests that p53 has a strong and selective inhibitory effect on the Rad51core promoter. Additional experiments overexpressing p53 in HeLa cells yielded similar results - supporting that this inhibitory effect is not limited to H1299 cells or p53 cells (Information S1). The p53 binding site is not required for tumor-selectivity of the Rad51 core promoter It has been reported that the Rad51core promoter contains p53 binding elements within the region 2159/2118 upstream of the transcription start [24]. To test the effect of this region on transcriptional activity of Rad51core, we derived a series of plasmid constructs, including (i) promoter truncations spanning this element (Figure 1D), and (ii) site-directed mutations or internal deletions of the binding sites (Figure 1E). We then inserted these constructs upstream of the promoterless luciferase reporter gene in our replication-defective Ad5 vector, and tested their transcrip- tional activity. We first tested promoter constructs that were truncated either immediately upstream of the putative p53 binding domain (Rad51-D161, which contains the p53 binding region) or immediately downstream of this element (Rad51-D117, which lacks the p53 binding region) (Figure 1D). These constructs were introduced into p53-null H1299 cells that were then transfected with the p53-GFP expression plasmid described above, and FACS-sorted into GFP+ (p53+) and GFP2 (p532) populations. The transcriptional activity of both of these Rad51 promoter elements was suppressed in p53-positive cells (GFP+ in Figure 4C) not in p53-negative cells (GFP2 in Figure 4C). Thus, the presence or absence of the p53 binding region within the Rad51 promoter (2161/2117) had no effect the ability of p53 to inhibit Rad51- mediated transcription. We next examined the transcriptional activity of site-directed mutants of the Rad51 promoter, targeting the p53 binding region. The Rad51mp53 construct is identical to the Rad51core promoter, except for mutations to the conserved bases of the putative p53 binding sites, while the Rad51Dp53 construct contains a precise deletion of the p53 binding region (2159/ 2118) (Figure 1E). Each of these constructs was introduced into a panel of normal and cancer cell lines, along with the Rad51core promoter construct. The results (Figure 3) showed that the transcriptional activity of the Rad51 core element was unaffected either by mutation (Rad51mp53) or deletion (Rad51dp53) of the p53 binding region even in cells that expressed functional p53. Based on these data, and the results presented in Figure 4C, we conclude that p53 suppresses the transcriptional activity of the Rad51 promoter through a mechanism that is independent of the reported p53 binding site [24] within the Rad51 promoter. Overexpression of p300 relieves p53 mediated repression of the Rad51core promoter p300 is a ubiquitous coactivator of transcription that has been reported to interact with many different transcription factors [25], including p53 [26]. We therefore hypothesized that p53 might indirectly affect Rad51 promoter activity through interactions with p300. To test this prediction, we transfected p53null H1299 cells PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e28714