Rad51 Targeted Adenoviral Vector Figure 4. p53 suppresses Rad51 promoter expression. (A) Cells with functional p53 have lower Rad51 promoter activity. 9 different cancer and non-cancer cell lines were grouped by their reported p53 status and transduced with Ad5DE1-Rad51full-luc vector at a MOI of 100. 24 hours post- infection, cells were lysed and luciferase activity of the cell lysates measured. Each point represents the mean value of independent experimental triplicates of a cell line. The median value of each group, indicated by the line, was determined to be significantly different by a Mann-Whitney analysis (p = 0.03). (B) Restoring expression of p53 in a p53-/- cell line reduces the activity of the Rad51 core promoter. H1299 cells were transfected with a plasmid encoding either a p53-GFP fusion protein or GFP flag. 12 hrs post-transfection, the cells were transduced with Ad5DE1-Rad51core-luc vector or Ad5DE1-CMV-luc at a MOI of 100 pfu/cell. 24 hrs thereafter, the cells were collected and sorted into GFP+ and GFP2 populations via FACS. The cells were then lysed and luciferase activity measured. Data are presented as mean values of independent experimental triplicates; error bars represent the standard deviation of the data value. The Rad51 promoter activity in p53 positive and negative cells was significantly different, as determined by a paired student’s T-test (p = 0.005); in contrast, the CMV promoter activity was statistically equivalent in both p53 positive and negative cells. (C) P53 mediated suppression of Rad51 promoter activity was not dependent on a reported p53 binding region (2161/2117). Methods were as described in (B), except that cells were transduced with Ad5DE1-Rad51D161-luc vector or Ad5DE1-Rad51D117-luc vector instead of Ad5DE1-Rad51core-luc (see Fig. 1D for schematic representations of these vectors). doi:10.1371/journal.pone.0028714.g004 with the pShuttle-Rad51core-luc plasmid along with increasing amounts of p300 expressing plasmid in the presence or absence of a plasmid expressing wild-type p53. The GFPflag expressing plasmid, pCMV-GFPflag, was used to as ‘‘filler’’ to ensure equivalent quantities of DNA were transfected into each sample. Successful transfection and expression of p53 and p300 was confirmed by western blot (Figure 5B). Consistent with the data Figure 5. Overexpression of p300/CBP overcomes p53-medi- ated repression of Rad51 core promoter activity. H1299 cells were cotransfected with a plasmid expressing luciferase under the control of the Rad51core promoter, in the presence or absence of plasmids encoding wild-type p53 or p300. Increasing amounts of the p300 expression were used (as indicated); the total amount of amount of transfected plasmid DNA in each reaction was maintained at a constant 4 mg by adding the necessary amount of irrelevant pCMV- GFPflag plasmid DNA. (A) 48 hours post transfection, cells were collected, lysed and transcriptional activity measured by Luciferase assay. Results shown represent the mean of independent experimental triplicates; error bars represent the standard deviation of the data. (B) Western blot analysis was performed to confirm expression of p53 and p300. 10 mg of total protein from each sample was separated on a 7.5% polyacrylamide gel, transferred to a nitrocellulose membrane, and probed for each respective protein using antibodies specific for the indicated proteins (p300, p53 and b-tubulin as a loading control). doi:10.1371/journal.pone.0028714.g005 presented in Figure 4, expression of p53wt repressed transcrip- tional activity from the Rad51core promoter. However, overex- pression of p300 relieved this p53 induced repression, in a dose dependent manner (Figure 5A). Moreover, when p300 was overexpressed at very high levels, Rad51core promoter activity was increased above basal levels (e.g., cotransfection of 2.0 mg of a p300 encoding plasmid increased transcriptional activity from the Rad51core promoter by 5.2-fold; Figure 5A). Tumor-selective induction of cell death, directed by the Rad51core promoter Having defined the core Rad51 promoter, and demonstrated its regulation by p53, we proceeded to examine whether this DNA element could be used to selectively drive expression of a cytotoxic effector protein in p53-defective tumor cells. We constructed an adenoviral vector encoding HSV thymidine kinase under the control of the Rad51core promoter (Ad5DE1-Rad51core-TK). A panel of p53 defective cancer cells and non-cancer cell lines were transduced with Ad5DE1-Rad51core-TK and an Ad5DE1-CMV- TK control vector at various moi (0 to 500 pfu/cell). Cells were simultaneously treated with ganciclovir (GCV) at concentrations ranging from 0 to 200 mM. The p53-defective cancer cells clearly demonstrated a GCV dose dependent loss of viability with both vectors (Figure 6), while the primary cell lines only showed loss of viability in cultures transduced with the control, CMV-driven, vector (Figure 7). This is exemplified by the fact that, at an MOI of 50 pfu/cell, Ad5DE1-Rad51core-TK caused almost 60% loss of viability in H1299 cells (Figure 6) treated with 1 mM GCV, while treatment of BJ fibroblasts (Figure 7) with 106 more vector and 2006 higher concentration of GCV resulted in less than 10% loss of viability. At these same doses, the control vector (Ad5DE1- CMV-TK) elicited in a similar ,60% loss of viability in H1299 cells, but a much greater, ,74% loss of viability, in BJ fibroblasts. Discussion Two previous attempts to map the minimal Rad51 promoter, both performed in the U2-OS osteosarcoma cell line, identified different essential promoter domains: 2204/258 [22] and 2536/ 2412 [23]. In this report, we performed a more comprehensive analysis of the Rad51 promoter in a broad range of cell lines, including both cancer cells and primary cells. Our experiments defined a small (447 bp) element from the Rad51 promoter that supports cancer specific transcriptional activity, and is sufficiently compact to allow its use in DNA delivery systems where transgene capacity is limited. We found no evidence for the presence of strong transcriptional suppressor or enhancer elements flanking this core element [22]. The broad range of cancers in which the core Rad51 promoter is active may be explained by its increased activity in cells lacking PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e28714