yielded compounds acting upstream of p53 to provoke p53 activation. This is illustrated by the fact that many of the identified drugs have also p53 independent functions to suppress tumor cell growth. Encouragingly, several of the compounds activate p53 without launching a cellular DNA damage response (i.e. do not impose genotoxic effects on the normal tissues), indicating that they employ activation of events other than DNA damage [19]. Given that p53 is a key activator of cell cycle arrest/apoptosis pathways, we considered that further development of small-molecule compounds inducing p53 is highly warranted and has potential for therapeutic exploitation. We demonstrate here successful implemen- tation of a cell-based high-content imaging screen to identify novel p53 pathway activating small-molecule lead compounds. This screen was devised to identify hits even in the presence of activation of the powerful p53-mediated apoptotic pathway. We present, both by genomic profiling and screening of a defined drug library, that the principal mean to activate p53 pathway is related to interference with DNA topology, likely by DNA intercalation. These approaches identify a substantial number of both new experimental lead molecules and drugs with well-known pharmacological profiles as potentially useful anticancer compounds. Materials and Methods p53 activity screen A cell-based assay for p53-dependent expression of a fluorescent reporter was established. A375 melanoma cells (ATCC CRL-1619) were stably transfected with a DsRed Express (Clontech) vector containing p53 consensus element [21] (p53DsRed reporter). p53DsRed reporter activation was verified by UVC, ionizing radiation (137Cs source) and nutlin-3 (Alexis Biochemicals) treatments. Cells were plated at 10,000 cells/well onto 96-well plates and treated at 3 mM (ChemDiv, Tripos) or 10 mM (Spectrum Collection) compound concentration for 24 h. Mock treatment (DMSO alone) and positive control (nutlin-3) was included in each 96-well plate. Following the incubation, the cells were fixed with paraformaldehyde (PFA) and nuclei were stained with Hoechst 33342 (Molecular Probes). The cells were imaged using Cellomics ArrayScan 4.5 high- content imaging platform (ThermoScientific) including Zeiss 200 M microscope with a 10x objective (Zeiss) and ORCA-AG CCD camera (Hamamatsu). Target Activation Bioapplication (Thermo- Scientific) algorithm was used for image acquisition and analysis of the total number of nuclei and percentage of p53DsRed reporter positive cells (responders). A minimum of 1700 cells/well was analyzed. Compounds inducing a minimum of two-fold increase of responders compared to mock were regarded as primary hits. All primary hits were additionally visually inspected for p53 reporter activation and DNA content. In the secondary screen the hits were tested over 0.1–10 mM concentration range. Compounds providing consistent p53DsRed reporter activation were selected for further testing. Z-factor [22] for the screen was 0.632. Viability assay Cells were plated in 96-well plates at a density of 10,000 cells/ well. Compounds were added in triplicate and the cells were incubated for three days. Cell viability was assayed using WST-1 cell proliferation reagent (Roche Diagnostics). The experiment was repeated two to three times and results are presented as % viability as compared to the control. Novel p53 Pathway Activators consent and as approved by the Johns Hopkins Medical Institute Institutional Review Board (J0002). Mononuclear cells were isolated from freshly harvested bone marrow aspirates, seeded at a density of 16106 cells/ml and incubated with the compounds for 24 hours. The compounds were removed and 56104 cells were plated and incubated for 14 days [23]. Colonies consisting of greater than 40 cells were counted. Immunoblotting analysis Cellular lysates were prepared and proteins were separated by SDS-PAGE. The following antibodies were used: DO-1 for p53, 2A10 for Hdm2, cH2AX (Upstate), p53Ser15 (Cell Signaling), KAP-1 (BD Transduction Laboratories), KAP-1 Ser824 (Bethyl Laboratories) and anti-p21 (Becton Dickinson). Horseradish peroxidase or biotin-streptavidin-horseradish peroxidase (Dako Cytomation) conjugated secondary antibodies were used. Equal protein loading was verified using Gapdh (p9.B.88, Europa Bioproducts Ltd). Immunofluorescence and quantitative image analysis Cells were grown on glass coverslips and fixed with PFA. cH2AX was detected with anti-H2AX (Ser139) antibody (Upstate) and Alexa488 conjugated secondary antibody (Molecular Probes), nuclei were stained with Hoechst 33342. Images were captured using Axioplan2 fluorescence microscopes (Zeiss) equipped with AxioCam HRc CCD-camera and AxioVision 4.5 software using EC Plan-Neofluar 40x/0.75 objective (Zeiss). p53 immunostaining for cells grown in 96-well plates was performed using DO-1 antibody and Alexa488-conjugate and nuclei were stained with Hoechst 33342. Images were acquired using ArrayScan 4.5 automated imaging platform and analyzed using Target Activa- tion Bioapplication algorithm (Thermo Scientific). The mean average intensity of $100 cells was quantified. Transcriptional profiling Cells were treated with the compounds or vehicle DMSO for six hours in four separate experiments and total RNA was isolated. Total RNA (1 mg) was reverse transcribed (Invitrogen SuperScript II reverse transcriptase); double strand cDNA was generated and column-purified (Affymetrix). Biotinylated cRNA was generated through in vitro transcription, fragmented and hybridized to GeneChip human U133A 2.0 arrays for 16 h at 45uC with constant rotation. Hybridized GeneChip was scanned using G3000 GeneArray Scanner (Affymetrix). Image analysis was performed using GeneChip Operating System 1.1.1 (GCOS) software (Affymetrix). Normalized data were imported into Partek Genomics Suite (Partek) software and followed with One-Way ANOVA analysis. Hierarchical clustering analysis was based on Euclidean distance metric and performed using R and Biocon- ductor software. Functional enrichment analysis Transcripts identified in the microarray were annotated for GO [24] assignments using DAVID database [25]. For enriched GO terms, P values from Fisher’s exact t-test were cut off at 0.01 on biological process level 4, and GO terms with more than 10 genes with an enrichment score higher than 2.5 were selected. For the KEGG pathways a P value ,0.05 and an enrichment score greater than 2 in a group of more than 10 genes were used. Human CFU-GM assay Aliquots of normal bone marrow were obtained from four normal allogenic bone marrow donors granting written informed Real-time qPCR Pooled total RNA (1 mg) was used to generate quantitative real- time PCR (qPCR) standard curve for all genes. Briefly, the pooled PLoS ONE | www.plosone.org 2 September 2010 | Volume 5 | Issue 9 | e12996