Novel p53 Pathway Activators Figure 4. Lead compounds have both DNA damage response-dependent and independent activities. A) A375 cells were treated with compounds BMH-7, -9, -15, -22 (5 mM), and BMH-21 (0.5 mM) for 8 hours and cell lysates were analyzed by immunoblotting for p53, p53Ser15, and cH2AX. The mean fold induction of p53Ser15, as compared to total p53, is shown below the blots (n = 2). All samples were normalized to Gapdh. B) Cells were treated with ATM inhibitor KU55933 (10 mM) for 90 min, followed by addition of BMH-7, -9, -15, -22 (5 mM), BMH-21 (0.5 mM), or ionizing radiation (IR) (10 Gy), and incubation for 6 hours. p53 stabilization was analyzed by immunoblotting. C) cH2AX foci formation of cells treated as in Figure 4B was analyzed by immunofluorescence. Nuclei were stained with Hoechst 33342. Scale bar, 10 mm. D) Cells were pretreated with KU55933 as in Figure 4B, followed by addition of the compounds for 30 min. KAP-1 and KAP-1 Ser824 was analyzed by immunoblotting. doi:10.1371/journal.pone.0012996.g004 compounds effectively reduced B cell lymphomagenesis in an orthotopic mouse model. Five of the compounds displayed striking effects by decreasing cell viability independently of p53. This underscores that the compounds in fact trigger upstream p53 activating events and activation of potent cytotoxic pathways. p53 is highly sensitive to DNA damage stress [2,32]. However, only two of the compounds (BMH-7 and BMH-15) activated ATM-dependent DNA damage signaling, i.e. acted as potentially genotoxic agents. The absence of the DNA damage response by four lead compounds indicates that their actions are unrelated to those directly causing DNA damage. The absence of DNA damage response may in fact be a preferable mechanism of action by potentially lowering normal cell toxicity, including genotoxicity. Notably, BMH-21, which had the most potent antitumorigenic activity, did not activate DNA damage signaling. Importantly, the improved screen protocol used here not only allowed selection of cell permeable compounds, but also facilitated the identification of lead molecules with antitumor activities in the nanomolar range. All compounds were found to intercalate with DNA. DNA is an extremely attractive target for cancer therapeutics. It poses exquisite sequence-specificity and unique structures at different metabolic states, and is also a widely targeted molecule by many PLoS ONE | www.plosone.org 7 September 2010 | Volume 5 | Issue 9 | e12996