Novel p53 Pathway Activators To further assess the regulation of known p53 target genes by the lead compounds we undertook qPCR analysis of eleven known p53 targets. In order to validate p53 dependency, a comparison between the responses in isogenic HCT116 p53+/+ and p532/2 cell lines [28] was conducted. The induction of the target genes by BMH-9, -21, -22, and -23 was clearly dependent on p53, whereas BMH-7 and BMH-15 had more restricted capacity to p53 dependent gene regulation (Figure 2C). These analyses show extensive activation of p53 downstream signaling by four lead compounds, and more limited by two. Novel lead compounds are antitumorigenic Given the prominent activation of the p53 responses, we analyzed the effects of the compounds on the viability of several tumor and normal cell lines. The lead compounds potently suppressed the viability of various tumor cells, albeit to different degrees (Figure 3A). These included cell lines wild-type, mutant and for p53, and showed that the antiproliferative activity of only BMH-9 was somewhat dependent on p53 (Figure S2). Comparison of the lead compound effects in three normal human cell lines and tumor cells indicated higher resistances of the normal cells to the compound antiproliferative activity (Table S3). We also assessed the potential toxicity of three representative compounds against human hema- topoietic progenitors. As shown in Figure 3B, BMH-9 and BMH-22 exhibited essentially no cytotoxicity and BMH-15 produced only a 10% inhibition of granulocyte-macrophage colony-forming units (CFU-GM). In contrast, topotecan, a TOP1 poison and known hematopoietic suppressor, had prominent toxicity. The results indicate that the lead compounds are potential novel anticancer agents without excessive toxicity in several human primary cells. Figure 3. Lead compounds are anti-tumorigenic. A) Tumor cell lines (A375, WM239, RPMI7951 melanoma, U2OS and SaOS2 osteosarcoma, BC3 lymphoma, and HCT116 colon adenocarcinoma cells) were incubated with BMH-7, -9, -15, -22, -23, nutlin-3 (10 mM), and BMH-21 (1 mM) in triplicate for three days. Cell viability was determined using WST-1 cell proliferation assay. Cell viability in control is set as 100%. Error bars represent SD. B) Dose-response curves of normal human CFU-GM. Shown is the clonogenic recovery of normal CFU-GM derived from four healthy individuals, treated with topotecan (TPT), BMH-9, BMH-15 and BMH-22. Error bars represent SD. C) BC3 cells were treated with BMH-15 and BMH-22 (10 mM) for 24 and 72 hours followed by flow cytometry. Cell cycle distribution and fraction of sub-G1 cells are indicated. D) NOD-SCID mice were injected i.p. with BC3luc cells, and tumors were allowed to establish for three days. Tumor take was confirmed by imaging and the mice were treated with i.p. injection of BMH-15 and BMH-22 (20 mg/kg) three times a week. Control animals received DMSO. The mice were imaged prior to each treatment and bioluminescence was quantified and plotted as total flux within a constant ROI. BMH-15 and BMH-22 caused significant reductions in tumor signals (ANOVA analyses, BMH-15, days 12, 14, 17, P values 0.0281, 0.0138, 0.0131, n = 5, respectively; BMH-22, days 12, 14, 17, P values 0.0259, 0.0160, 0.0045, n = 4, respectively; mock, n = 5). Error bars, SD. E) Bioluminescence images of the mice at day 8 and day 17. Heatbar indicates the bioluminescence intensities. doi:10.1371/journal.pone.0012996.g003 PLoS ONE | www.plosone.org 5 September 2010 | Volume 5 | Issue 9 | e12996