equally efficient in mediating ganciclovir-dependent killing of p53 defective cancer cells. This demonstrates the robust ability of the Rad51 promoter to drive expression of a cytotoxic transgene in cancer cells. Moreover the Ad5DE1-Rad51core-TK vector was not cytotoxic to normal cell lines, whereas the Ad5DE1-CMV-TK vector elicited GCV-dependent cell death even in normal cells. The toxicity of the CMV-driven vector demonstrates that adenoviral vectors can efficiently transduce normal cells, under- scoring the selectivity and potential utility of the Rad51 promoter. The efficacy with which the vector killed p53-defective tumor cells was correlated with the transcriptional activity of the Rad51core promoter in the various cells. Overall, these data establish proof of principle support for the use of the Rad51core promoter in virally vectored gene therapies for p53 defective cancers. Materials and Methods Cell culture HEK 293A [41], Wi-38 [42], HCA-2 [43], Hela [44], BJ [45], MCF-7 [46], A549 [46] and MRC-5 [47] cells were cultured in Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% FBS (Gibco) and 16 Pen Strep Glutamine (Gibco). H1299 [48], BxPc-3 [31], U251 [46], SF-539 [46] cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS (Gibco) and 16 Pen Strep Glutamine (Gibco). SAEC (Lonza) and nHeps (Lonza) cells were cultured in the growth media suggested by the vendor. Cloning of Rad51 promoters The Rad51full promoter was PCR amplified from human genomic DNA and cloned into peGFP (Clontech) as described [18]. All subsequent truncations were made by PCR or restriction enzyme digestion from peGFP-Rad51full. Rad51-mp53 and Rad51-Dp53 were commercially synthesized by GeneArt (Regens- burg, Germany). Rad51-mp53 is derived from Rad51core and has the underlined nucleotides in the region 2159/2118 mutated from AAACTCGCGCAGGATCAAGCTCTCGAGCTCCCG- TCTTGGGT to AAAGGCCCGCAGGATGGCCCTCTCGA- GCTCCCGTGGCCGGT. Rad51-Dp53 is also derived from Rad51core but has the entire region deleted. Virus construction All adenovirus vectors were made with the AdEasyTM system (Stratagene). Rad51 promoter constructs were cloned upstream of a luciferase cassette in a pShuttle backbone, recombined with pAdEasy and linearized to form an adenoviral genome. This DNA was transfected into HEK 293A cells for virus production and amplification. Viruses were collected, purified on a CsCl gradient and titered by plaque assay. A control vector expressing luciferase and GFP from the constitutive CMV promoter, Ad5DE1-CMV- GFPluc, was created by cloning the luciferase gene from pGL3- basic into the pAdtrack shuttle plasmid and recombined into pAdeasy; virus production was then completed as described above. A control vector that expressed only luciferase under the control of the CMV promoter, Ad5DE1-CMV-luc, was purchased from Vector Biolabs (Philadelphia, PA). Measuring promoter activity Each cell type was seeded at a density of 56105 cells per well into 6-well plates and cultured overnight. The following morning, the cell culture media was removed and replaced with fresh media containing the indicated adenoviral vectors at a multiplicity of infection (MOI) of 100 pfu/cell. 24 hours after transduction the cells were collected and lysed with Passive Lysis Buffer (Promega). Rad51 Targeted Adenoviral Vector Luciferase assays were performed with Luciferase Assay System from Promega and luminescence measured on a DTX880 multimode plate reader (Beckman Coulter). All samples were normalized by total protein content as determined by Bradford assay. p53 rescue experiment 16106 H1299 cells were seeded in 30 mm cell culture plates and cultured overnight. The following morning, 4.0 mg of either pEGFP-p53 or pCMV-GFPflag was transfected into the cells using Lipofectamine2000 (Invitrogen). 12 hrs post-transfection, the cells were infected with Ad5DE1-Rad51core-luc or Ad5DE1-CMV-luc at an MOI of 100. 24 hrs post-infection, the cells were collected and sorted into GFP+ and GFP2 populations on a FACSAriaII cell sorter. The cells were then lysed and luciferase activity measured with the Luciferase Assay System from Promega, described above. The experiment presented in Information S1, using HeLa cells, was performed using essentially methods. Co-activator overexpression experiments H1299 cells were seeded in 6-well plates at a density of 2.56105 cells/well. The following day cells were cotransfected with a plasmid expressing luciferase under the control of Rad51core promoter (1 mg pShuttle-Rad51core-luc), in the presence or absence of plasmids expressing p53wt (1 mg pCMV-p53wt), p300 (0.5–2 mg pCMV-p300) and GFPflag (pCMV-GFPflag) using Lipofectamine2000 (Invitrogen). The amount of irrelevant GFPflag plasmid (‘‘filler’’) DNA was adjusted to maintain a total of 4 mg transfected DNA in all conditions. 6 hours post transfection, the serum free transfection medium was replaced with fresh culture medium and cell were then incubated for 48 hours at 37u prior to collection, lysis and performance of luciferase assays, as described above. GCV/HSVtk treatment and cell viability measurement Cells were seeded in 96-well plates at a density of 16103 cells per well. 24 hours later, culture media was aspirated and replaced with fresh media containing appropriate dilutions of ganciclovir and vector. The cells were returned to incubate at 37u C for 5 days after which cells were stained with AlamarBlueTM (Invitrogen). Cell culture media was removed from cells, replaced with fresh media + AlamarBlueTM, and cells were then incubated at 37uC for an additional 12 hours. Fluorescence (EX = 535 nm, EM = 595 nm) was measured in a DTX880 multimode plate reader (Beckman Coulter). Cell viability was calculated as a percentage of the mean value of untreated cells. Supporting Information Information S1 Rad51core promoter activity correlates with host cell expression of Rad51 protein. (A) Nuclear extracts were isolated from each cell type and 3 mg of total protein was separated on a 10% polyacrylamide gel, transferred to a nitrocellulose membrane, and probed with a monoclonal antibody for Rad51. The blots were stripped and re-probed for b-tubulin as a loading control. All displayed samples were run on the same blot. (B) Rad51 expression was quantitated by densitometry from the blot shown in panel (A) using QuantityOne software (BioRad); Rad51 expression was then normalized in terms of housekeeping protein levels (b-tubulin). Rad51 expression levels in normal fibroblasts (Wi-38 cells) were defined as 1, and Rad51 expression levels in the other cells were then expressed relative to this. Shown is a scatterplot of endogenous Rad51 protein content for each cell line, versus the level of Rad51core promoter activity in the same PLoS ONE | www.plosone.org 10 December 2011 | Volume 6 | Issue 12 | e28714