Mismatch Repair Protein Deficiency Compromises Cisplatin-induced Apoptotic Signaling

Mismatch repair (MMR) proteins participate in cytotoxicity induced by certain DNA damage-inducing agents, including cisplatin (cis-diamminedichloroplatinum(II), CDDP), a cancer chemotherapeutic drug utilized clinically to treat a variety of malignancies. MMR proteins have been demonstrated to bind to CDDP-DNA adducts and initiate MMR protein-dependent cell death in cells treated with CDDP; however, the molecular events underlying this death remain unclear. As MMR proteins have been suggested to be important in clinical responses to CDDP, a clear understanding of MMR protein-dependent, CDDP-induced cell death is critical. In this report, we demonstrate MMR protein-dependent relocalization of cytochrome c to the cytoplasm and cleavage of caspase-9, caspase-3, and poly(ADP-ribose) polymerase upon treatment of cells with CDDP. Chemical inhibition of caspases specifically attenuates CDDP/MMR protein-dependent cytotoxicity, suggesting that a caspase-dependent signaling mechanism is required for the execution of this cell death. p53 protein levels were up-regulated independently of MMR protein status, suggesting that p53 is not a mediator of MMR-dependent, CDDP-induced death. This work is the first indication of a required signaling mechanism in CDDP-induced, MMR protein-dependent cytotoxicity, which can be uncoupled from other CDDP response pathways, and defines a critical contribution of MMR proteins to the control of cell death.The MMR² system of proteins plays roles in diverse cellular processes, perhaps most notably in preserving genomic integrity by recognizing and facilitating the repair of post-DNA replication base pairing errors. Recognition of these errors and recruitment of repair machinery is performed by the MutSα complex (consisting of the MMR proteins MSH2 and MSH6) or MutSβ complex (consisting of MSH2 and MSH3). Defects in MMR proteins render cells hypermutable and promote microsatellite instability, a hallmark of MMR defects. MMR protein defects are found in a wide variety of sporadic cancers, as well as in hereditary non-polyposis colorectal cancer (1).In addition to their role in DNA repair, MMR proteins also play a role in cytotoxicity induced by specific types of DNA-damaging chemotherapeutic drugs, such as CDDP, which is utilized clinically to treat a number of different cancer types. MutSα recognizes multiple types of DNA damage, including 1,2-intrastrand CDDP adducts and O⁶-methylguanine lesions (2). Treatment of cells with compounds that induce these types of lesions, including CDDP and methylating agents such as N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), results in MMR protein-dependent cell cycle arrest and cell death (3–7). This suggests that MMR proteins, in addition to their role in DNA repair, are also capable of initiating cell death in response to certain types of DNA damage.Cells treated with DNA-damaging agents frequently activate an apoptotic cell death pathway mediated by the mitochondria. This intrinsic death signaling pathway predominantly involves the coordinated activity of two groups of proteins: pro-death members of the Bcl-2 family that control the integrity of mitochondrial membranes, and members of the caspase family of cysteinyl proteases that proteolytically cleave intracellular substrates, giving rise to apoptotic morphology and destruction of the cell (8, 9). Pro-death Bcl-2 family members, such as Bax and Bak, target the outer mitochondrial membrane and cause the cytosolic release of pro-death factors residing within the mitochondria of unstressed cells (8). Predominant among these factors is cytochrome c, whose cytoplasmic localization results in the formation of a caspase-activating platform known as the apoptosome (10). This complex includes the adaptor protein Apaf-1, and when formed the apoptosome promotes the cleavage and activation of caspase-9 (11, 12). Once activated, this apical caspase proceeds to cleave and activate caspase-3, the predominant effector protease of apoptosis.A significant amount of evidence has been gathered illustrating MMR protein-dependent pro-death signaling in response to methylating agents (13–16, 3). In contrast, the MMR protein-dependent cytotoxic response to CDDP is largely unknown, with only the p53-related transactivator protein p73 and the c-Abl kinase clearly implicated as potential mediators of CDDP/MMR protein-dependent cell death in human cells (17, 18). Interestingly, ATM, Chk1, Chk2, and p53, which are activated in an MMR protein-dependent manner after treatment of cells with MNNG (3, 13), are not involved in the MMR-dependent response to CDDP (7, 17). In addition, the magnitude of MMR protein-dependent cell death induced by methylating agents and CDDP differs (4). These findings suggest that unique signaling pathways may be engaged by MMR proteins depending upon the type of recognized lesion. As such, there is a requirement for further study of the molecular events underlying MMR protein-dependent cell death and cell cycle arrest for each type of recognized DNA lesion. This is particularly relevant in the case of CDDP, as evidence from a limited number of retrospective clinical studies suggests that MMR proteins play an important role in patient response to CDDP. Several studies examining immunohistochemical staining against MSH2 or MLH1 have demonstrated that levels of these proteins are reduced in ovarian and esophageal tumor samples following CDDP-based chemotherapy (19, 20). Low levels of MMR protein post-chemotherapy seem to be predictive of lower overall survival in a certain subset of tumors (esophageal cancer), but not others (ovarian and non-small cell lung cancer) (19–21). Two recent studies examining MMR protein levels and microsatellite instability in germ cell tumors from patients receiving platinum-based chemotherapy have suggested a prognostic value for pre-chemotherapy MMR protein status in these tumors (22, 23). This potential clinical relevance underscores the need for a greater understanding of MMR protein-dependent mechanisms of CDDP-induced cell death.In this study, we report that CDDP induces an MMR protein-dependent decrease in cell viability and MMR protein-dependent signaling in the form of cytochrome c release to the cytoplasm and cleavage of caspase-9, caspase-3, and PARP. Chemical inhibition of caspases specifically attenuates CDDP/MMR protein-dependent loss of cell viability, indicating a requirement for caspase activation in this process and uncoupling MMR protein-dependent cytotoxic signaling from other CDDP response pathways. Additionally, the CDDP-induced, MMR protein-dependent cytotoxic response is independent of p53 signaling. Our results demonstrate for the first time an MMR protein-dependent pro-death signaling pathway in cells treated with CDDP.