Novel p53 Pathway Activators Figure 5. Transcriptional profiling and gene expression analysis of the lead compounds. A) MCF-7 cells were treated with BMH-7, -9 (10 mM), BMH-15, -22, -23 (5 mM), and BMH-21 (0.5 mM) for 6 hours, and control cultures were mock-treated with DMSO. Total RNA was isolated from four independent experiments. Differentially expressed transcripts (P,0.01, total of 5926 transcripts) were identified by One-Way ANOVA analysis and hierarchical clustering was performed using R software. Dendrograms and heat map values are shown. B) Venn diagrams of transcripts shared by BMH-9/22/23 and BMH-7/15/21. C) Hierarchical clustering of 118 target genes shared by all compounds. D) KEGG pathways. Pathways extensively shared are shown (P,0.05, enrichment score .2.0, over 10 targets/category). E) qPCR verification of transcripts identified by profiling. qPCR was performed on randomly selected eight up-regulated and ten down-regulated genes. Fold change as compared to GAPDH is shown. Note that CYP1A1 bars for BMH-21, -22, -23 are truncated (actual values 55.2, 10.1, and 6.2, respectively). Data represent duplicate biological experiments and duplicate qPCR reactions. Error bars represent SD. doi:10.1371/journal.pone.0012996.g005 anticancer drugs [37,38]. These interact with DNA typically through three modalities, namely DNA intercalation, groove binding and covalent interactions causing cytotoxicity and therapeutic advantage [30,38–42]. DNA intercalators, beyond those that induce DNA damage, lead to local structural changes in DNA, including unwinding and lengthening of the DNA helix [30,37,38]. These events may lead to alterations in DNA metabolism, and halter transcription and replication, and induce p53 [43]. Interestingly however, the lead compounds cause distinct transcriptional programming, which includes both transcriptional induction and repression, and are consistent with activation of pathways regulating cell cycle progression, p53 pathway, ubiqui- tin-dependent proteolysis and DNA damage surveillance (Figure 5). These findings indicate that the compounds do not cause a general transcriptional repression, but rather, selective regulation. Fur- thermore, this transcriptional programming seems to be largely shared by the lead compounds, especially those by BMH-9, BMH- 22 and BMH-23. The implication of these findings is that the compounds may also selectively alter DNA metabolism in a manner that may be dependent on the DNA sequence, conformation or interactions of the compounds with chromatin proteins. The high frequency of quinoline-derivatives among the lead compound profiles and the chemical screens, and their known property of DNA intercalation [39], raises the possibility that this property may predispose to p53 induction or antitumor activity. Many of the agents identified by the chemical screens or lead profiling analyses have recognized toxicities and some, carcino- genic effects. Therefore, activation of p53 may reflect severe cellular stress that may culminate in the elimination of tumor cells or, in susceptible normal cells, toxicity. However, the novel lead compounds had low to non-existent human bone marrow toxicity and no in vivo toxicity in mouse. Considering their favorable pharmacokinetic predictors and demonstrated antitumor activity we propose that they represent promising novel anticancer lead compounds. PLoS ONE | www.plosone.org 8 September 2010 | Volume 5 | Issue 9 | e12996