Oncogene (2010) 29, 6149–6159 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc ORIGINAL ARTICLE Status of p53 in human cancer cells does not predict efficacy of CHK1 kinase inhibitors combined with chemotherapeutic agents S Zenvirt1, N Kravchenko-Balasha1 and A Levitzki Unit of Cellular Signaling, Department of Biological Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel DNA damage checkpoints cause cell cycle arrest, allowing Millar, 2006). An important regulator of this response DNA repair before resumption of the cell cycle. These is the tumor suppressor p53. In response to DNA checkpoints can be activated through several signaling damage, p53 is activated and stabilized by the Ser/Thr pathways. Checkpoint activators include p53, checkpoint kinases Ataxia-Telangiectasia mutated and Ataxia- kinase 1 (CHK1), checkpoint kinase 2 and/or MAPKAP Telangiectasia- and RAD3-related (Oren, 2003; Meek, kinase 2 (MK2). Many cancer cells lack p53 activity and, 2004; Harris and Levine, 2005). The activated p53 therefore, depend on alternative checkpoint activators to protein stimulates the expression of p21, which inhibits arrest the cell cycle following DNA damage. Inhibition of the cyclin-dependent kinases, resulting in cell cycle arrest these pathways is expected to specifically sensitize these at both the G1-to-S (G1/S) and the G2-to-M (G2/M) p53-deficient cells to DNA damage caused by chemother- transitions (Vogelstein et al., 2000). In the absence of apy. Using isogenic p53-proficient and p53-deficient cancer p53, the G1/S checkpoint cannot be properly activated. cell lines, we show that inactivation of CHK1, but not Intra-S and G2/M checkpoints can be activated through MK2, abrogates cell cycle arrest following chemotherapy, the Ser/Thr kinases checkpoint kinase 1 (CHK1), specifically in p53-deficient cells. However, we show that checkpoint kinase 2 (CHK2) and MAPKAP kinase 2 CHK1 is required to maintain genome integrity and cell (MK2). CHK1 is activated by Ataxia-Telangiectasia- viability, and that p53-proficient cells are no less sensitive and RAD3-related following genotoxic stress, CHK2 is than p53-deficient cells to CHK1 inhibition in the pre- activated by Ataxia-Telangiectasia mutated following sence of DNA damage. Thus, combining CHK1 inhibition double strand DNA breakage such as that caused by with DNA damage does not lead to preferential killing ionizing radiation and MK2 is activated by the p38 of p53-deficient over p53-proficient cells, and inhibiting MAP kinase following ultra-violet radiation or che- CHK1 does not appear to be a promising approach for motherapy (Abraham, 2001; Bartek and Lukas, 2003; potentiation of cancer chemotherapy. Shiloh, 2003; Manke et al., 2005; Karlsson-Rosenthal Oncogene (2010) 29, 6149–6159; doi:10.1038/onc.2010.343; and Millar, 2006; Reinhardt et al., 2007). CHK1, CHK2 published online 23 August 2010 and MK2 phosphorylate and thereby inactivate the Cdc25 phosphatases, resulting in inactivation of cyclin- Keywords: CHK1; checkpoint; cancer; p53 dependent kinases and cell cycle arrest (Donzelli and Draetta, 2003; Karlsson-Rosenthal and Millar, 2006). In about half of human cancers, the activity of p53 is Introduction compromised and the G1/S checkpoint fails (Vogelstein et al., 2000; Reinhardt et al., 2007). It has been suggested DNA damaging agents have been in use for cancer that CHK1 or MK2 inhibitors would abrogate the therapy for decades. It has been suggested that these remaining checkpoints in cancer cells lacking functional agents could be potentiated by inhibition of the DNA p53 and this would lead to preferential sensitization of damage checkpoints (Zhou et al., 2003; Kawabe, 2004; these cancer cells to chemotherapy over normal p53- Zhou and Bartek, 2004; Stark and Taylor, 2006; proficient cells (Zhou et al., 2003; Kawabe, 2004; Zhou O’Connor et al., 2007; Ashwell et al., 2008; Bucher and Bartek, 2004; Stark and Taylor, 2006; O’Connor and Britten, 2008; Lieberman, 2008). In response to et al., 2007; Ashwell et al., 2008; Bucher and Britten, DNA damage, the cell cycle is arrested at specific 2008; Lieberman, 2008). A recent publication advocated checkpoints, allowing DNA repair or, if the damage the utilization of MK2 inhibitors, and reported that the cannot be repaired, activation of programmed cell death G2/M checkpoint in p53À/À ras transformed cells (Zhou and Elledge, 2000; Karlsson-Rosenthal and required MK2 (Reinhardt et al., 2007). In view of these reports concerning the utility of CHK1 and/or MK2 Correspondence: Professor A Levitzki, Department of Biological inhibitors as sensitizers of p53À/À cells to chemother- Chemistry, Unit of Cellular Signaling, The Hebrew University of apy, we examined the sensitivity of two isogenic pairs of Jerusalem, Givat Ram, Jerusalem, Israel 91904, Israel. p53-proficient and p53-deficient cancer cell lines to E-mail: [email protected] DNA damaging agents, when CHK1 or MK2 expres- 1These authors contributed equally to this work. Received 20 January 2010; revised and accepted 6 July 2010; published sion was knocked down by small interference RNA online 23 August 2010 (siRNA) or when their catalytic activity was inhibited. p53, CHK1 and chemotherapy S Zenvirt et al 6150 We discuss our results in view of the published literature nucleation were also detected (Figure 2bV). When no and question whether there is wide utility for CHK1 or chemotherapeutic drug was administered, 5–8% of the MK2 inhibitors as anti-cancer agents. cells that were transfected with non-targeting siRNA were in mitosis in both the cell lines (Figure 2c). Knocking down CHK1 in p53-proficient U2OS cells Results resulted in a decrease in mitosis, but not in p53-deficient cells. Knockdown of MK2 had no effect in either cell CHK1 but not MK2 is responsible for cell cycle arrest line. When CHK1 and MK2 were knocked down of p53 deficient cells in response to chemotherapy together, knockdown of MK2 antagonized the effect We first examined which checkpoint kinase should be of CHK1 knockdown in the p53-proficient cells inhibited in order to specifically abrogate checkpoints in (Figure 2c). p53-deficient cancer cells undergoing chemotherapy. We DNA damage is expected to activate checkpoints, used siRNA to inhibit the expression of CHK1, MK2 or resulting in cell cycle arrest. As expected, exposure of both. To verify the role of p53 in the maintenance of the cells that were transfected with non-targeting siRNA to checkpoint, we used a pair of isogenic cell lines derived chemotherapeutic drugs dramatically reduced the num- from U2OS: U2OS-LacZÀ, a p53-proficient cell line ber of mitotic cells in both the p53-proficient and p53- stably expressing siRNA against LacZ, and U2OS-p53À, deficient U2OS cell lines, indicating the onset of cell which stably expresses siRNA against p53 (Raver- cycle arrest. In most of these arrested cells, cyclin B1 Shapira et al., 2007). To confirm that U2OS-p53À have accumulated in the cytoplasm, indicating that they were abrogated activity of p53, we measured the effect of arrested at G2 or S phase (Figure 2bIV). Inhibition of Nutlin (Mdm2 inhibitor) on induction of p53-regulated the kinases that are responsible for the onset of the genes. As shown in Figure 1a, Nutlin induced the checkpoints is expected to abrogate the cell cycle arrest expression of p21 under basal conditions as well as induced by DNA damage. We indeed found that under cisplatin (CDDP) treatment in U2OS-LacZÀ cells, knocking down CHK1 restored mitosis in the p53- whereas no induction occurred in U2OS-p53À cells. deficient cells, but not in the p53-proficient cells, Moreover, under CDDP treatment, U2OS-LacZÀ cells following treatment with CDDP or irinotecan underwent caspase-3 dependent apoptotic cell death, (Figure 2c). Thr3-phosphorylation of histone H3 is a known to be p53 mediated (Figure 1b), whereas U2OS- marker of cells entering prophase (Dai et al., 2005). p53– cells did not activate caspase-3, as expected from When cells were knocked down for CHK1 and treated cells lacking p53. with CDDP, U20S-LacZ– (p53-proficient) cells were Western blot analysis confirmed that efficient and arrested before prophase, but U20S-p53À (p53-deficient) specific knockdown of CHK1 or MK2 was achieved cells had an increase in phosphorylated H3, indicating within 48 h of transfection and lasted for at least 3 days that they were able to proceed into prophase (Figure 2a and data not shown). One day post siRNA (Figure 2d). transfection, cells were exposed to the DNA cross-linker These results indicate that CHK1 is responsible for agent CDDP, or the topoisimerase I inhibitor irinote- the chemotherapy-induced cell cycle arrest in the can, both at concentrations of 10 mM. absence of p53. In contrast, knocking down MK2 did To detect checkpoint activation and cell cycle arrest, not affect the number of cells undergoing mitosis in we used immuno-fluorescence staining for cyclin B1 and either cell line, independent of the status of p53. These 40,6-diamidino-2-phenylindole staining for heterochro- results clearly indicate that CHK1, but not MK2, is matin, 24 h after application of the chemotherapeutic required for the activation of the DNA damage drugs. Cells that had entered mitosis were identified by checkpoint following chemotherapy in these cells. In the nuclear staining of cyclin B1 and by chromosome addition, the combined knockdown of CHK1 and MK2 condensation (Figure 2bII). Cells at later stages of had a smaller effect than the knockdown of CHK1 alone mitosis were identified by the morphology of the (Figure 2c), suggesting that MK2 knockdown is dividing chromatin (Figure 2bIII). Cells with micro- antagonistic to CHK1 knockdown. No No chemotherapy Cisplatin chemotherapy Cisplatin - - - - U2OS: -LacZ- -p53- -LacZ- -p53- U2OS: -LacZ -p53 -LacZ -p53 -N +N -N +N -N +N -N +N p53 p53 p21 p21 p19 - CASP3 p17 - Tubulin Tubulin Figure 1 Abrogated activity of p53 signaling in U2OS-p53À cells.