1935 Differential roles of checkpoint kinase 1, checkpoint kinase 2, and mitogen-activated protein kinase–activated protein kinase 2 in mediating DNA damage–induced cell cycle arrest: implications for cancer therapy Zhan Xiao, John Xue, Thomas J. Sowin, MK2-deficient cells, Cdc25A protein, which is critically and Haiying Zhang required for the mitotic progression following checkpoint abrogation, becomes greatly depleted. In summary, our Cancer Research, Abbott Laboratories, Abbott Park, Illinois findings show that Chk1 is the only relevant checkpoint kinase as a cancer drug target and inhibition of other Abstract checkpoint kinases in addition to Chk1 would be nonpro- ductive. [Mol Cancer Ther 2006;5(8):1935–43] Mammalian cells initiate cell cycle arrest at different phases of the cell cycle in response to various forms of genotoxic stress to allow time for DNA repair, and thus Introduction preserving their genomic integrity. The protein kinases DNA-targeted agents are among the most effective in checkpoint kinase 1 (Chk1), checkpoint kinase 2 (Chk2), clinical cancer therapy and constitute the cornerstones of and mitogen-activated protein kinase–activated protein modern cancer treatment. These agents can be divided into kinase 2 (MK2) have all been shown to be involved in cell four main classes: alkylating agents, antimetabolites, top- cycle checkpoint control. Recently, cell cycle checkpoint oisomerase inhibitors, and radiomimetics. Their exact abrogation has been proposed as one way to sensitize mechanisms of action vary greatly; however, a common cancer cells to DNA-damaging agents due to the expected theme is that they all confer DNA damage, directly or induction of mitotic catastrophe. Due to their overlapping indirectly, and induce cell cycle checkpoints. Despite their substrate spectra and redundant functions, it is still not effectiveness at elevating the overall survival rates of cancer clear which kinase is mainly responsible for the cell cycle patients, they have serious intrinsic limitations such as arrests conferred by clinically relevant chemotherapeutics. widespread tumor-resistance and severe toxicity in normal Thus, the issue remains about which kinase is the most tissues, resulting in a narrowtherapeutic window(1). therapeutically relevant target and, more importantly, Mammalian cells have established highly elaborate sur- whether multiple kinases might need to be targeted to veillance systems to detect DNA damages and other forms of achieve the best efficacy in light of recent studies showing genotoxic stress, which is essential to maintain the genomic superior efficacy for pan-receptor tyrosine kinase inhib- integrity and, hence, cellular viability. When damage is itors. To clarify this issue, we investigated the roles of the detected, cells activate sophisticated pathways, called cell three kinases in response to different genotoxic stresses cycle checkpoints, to arrest cells in different phases of the cell through small interfering RNA–mediated specific target cycle to allowsufficient time for DNA repair. In normal cells, knockdowns. Our result showed that only the down- checkpoint responses are a critical safeguard to prevent regulation of Chk1, but not of Chk2 or MK2, abrogated tumorigenesis promoted by genetic instability; however, camptothecin- or 5-fluorouracil–induced S-phase arrest or in tumor cells, checkpoints constitute a major mechanism doxorubicin-induced G2-phase arrest. This was followed of resistance to chemotherapeutic drugs that damage DNA by mitotic catastrophe and apoptosis. Moreover, double because they reduce the effects of these drugs (2, 3). inhibition of Chk1 and Chk2 failed to achieve better It has previously been postulated that targeting the efficacy than Chk1 inhibition alone; surprisingly, inhibition cellular checkpoint pathway could be an attractive ap- of MK2, in addition to Chk1 suppression, partially reversed proach to circumvent the cancer resistance as discussed the checkpoint abrogation and negated mitotic catastro- above. The rationale is that in normal cells, DNA damage phe. We further showed that this is due to the fact that in would arrest the cells mostly in G1 in a p53-dependent manner, whereas p53-deficient tumors, accounting for over half of all tumor-types, have to rely on the checkpoint Received 2/9/06; revised 4/11/06; accepted 5/31/06. mediators to arrest cells at S or G2-M checkpoint. Therefore, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked checkpoint inhibition would only abrogate the cell cycle advertisement in accordance with 18 U.S.C. Section 1734 solelyto blocks in tumor cells to induce mitotic catastrophe and indicate this fact. apoptosis, but mostly spare the normal cells. This may offer Requests for reprints: Haiying Zhang, Cancer Research, Abbott a potential feasible therapeutic window (4, 5). Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064-6101. Phone: 847-938-4857; Fax: 847-935-7551. Two structurally unrelated but functionally similar E-mail: [email protected] protein serine/threonine kinases, checkpoint kinase 1 Copyright C 2006 American Association for Cancer Research. (Chk1) and checkpoint kinase 2 (Chk2), have emerged as doi:10.1158/1535-7163.MCT-06-0077 the major mediators of cell cycle checkpoints in response Mol Cancer Ther 2006;5(8). August 2006 Downloaded from mct.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. 1936 Roles of Chk1, Chk2, and MK2 in Cell Cycle Arrest to genotoxic stress. Homozygous Chk1 knockout is lethal in Mitogen-activated protein kinase–activated protein ki- mouse embryonic stem cells. The mouse embryonic stem nase-2 (MK2) was discovered as a protein kinase activated cells that conditionally lack the Chk1 gene cannot prevent by extracellular signal–regulated kinase 1/2 from rabbit mitotic entry in response to ionizing radiation, showing skeletal muscle. Extracellular signal–regulated kinase 1/2 that Chk1 is required for the G2-M checkpoint (6). In adult and p38 were first reported to activate MK2 and MK3 somatic cells, Chk1 deficiency does not result in lethality in vitro, but it was later found that extracellular signal– and cells are viable and display normal cell cycle profiles regulated kinase 1 and 2 are not physiologic kinases for (7, 8). Complete deficiency of Chk1 in avian DT-40 MK2. Rather, MK2 activity is potently stimulated by h lymphoma cells abolished DNA damage–induced G2 various activators of p38 and p38 (19, 20). The targeted arrest and undermined S-phase checkpoint in response to deletion of the MK2 gene in mice provided the unexpected replication stress (8). Small interfering RNA (siRNA)– result that although p38 mediates the activation of many mediated knockdown of Chk1 in various human cancer cell similar kinases, MK2 seems to be the key kinase responsible lines also revealed an essential role of this kinase in both S for p38-dependent biological processes involving cytokine and G2-M DNA damage checkpoints (9, 10). Unlike Chk1, synthesis (21). Loss of MK2 leads to a defect in lipopoly- Chk2 knockout mice are viable and fertile (11). Although saccharide-induced synthesis of cytokines such as TNF-a structurally distinct from Chk1, Chk2 shares overlapping and g-IFN. Consistent with a role for MK2 in inflammatory substrate specificity with Chk1 and can phosphorylate responses, MK2-deficient mice showincreased susceptibil- critical Chk1 substrates, such as Cdc25A and Cdc25C, ity to infection (22). A recent finding has added new both in vitro and in vivo. Additionally, Chk2 is rapidly complexities to the above paradigm. Manke et al. showed phosphorylated and activated following exposure to that MK2 recognizes the same phosphorylation sites on various DNA-damaging agents such as ionizing radiation Cdc25B/C as Chk1/Chk2. More importantly, MK2 is or topoisomerase inhibitors. This indicated that Chk2 also directly responsible for Cdc25B/C phosphorylation and plays a role in cell cycle checkpoints (12). Consistently, their subsequent 14-3-3 binding in response to UV-induced studies with dominant-negative Chk2, siRNA-mediated DNA damage in mammalian cells. Down-regulation of Chk2 ablation, or intrinsic cellular Chk2 deficiency have all MK2 eliminates DNA damage–induced G2-M and intra-S confirmed a role of Chk2 in the S and G2 checkpoints in phase checkpoints. Therefore, it was proposed that MK2 response to double-strand breaks in various immortalized is a newmember of the DNA damage checkpoint kinase human cell types (13–15). In contrast to these reports family that functions in parallel with Chk1 and Chk2 to showing that Chk2 is required for checkpoint induction in integrate DNA damage signaling responses and cell cycle human cell lines, murine fibroblasts with Chk2 deletion arrest in mammalian cells (23, 24). showed no significant defects in the S-phase checkpoints, Judging from these results, it is still far from clear which indicating that it is not a major checkpoint mediator in checkpoint kinase is the major mediator of cell cycle arrest mice (11, 16, 17). This discrepancy was attributed to the in response to various DNA-damaging agents, especially differential requirements of Chk2 between human and the clinically relevant ones. It is possible that different murine systems or to the slowproliferation rate of murine forms of DNA damage require different checkpoint fibroblasts versus the fast growth rate of immortalized kinases to enforce the arrest.