MEF forfigure 2were treated with a single dose of 5 Gy at an exposure rate of 2.44 Gy/min and lysed in lysis buffer 2 h later. caretakers prevent DNA damage or facilitate its repair (2). DNA damage checkpoint machineries monitor the genome for problems that adversely affect DNA replication or mitosis, and halt cell cycle progression to allow time for repair. If the damage is severe or irreparable, these machineries engage either cell death Epoxomicin (apoptosis) or permanent cell cycle arrest (cellular senescence) pathways. These cellular damage responses PRKCZ are crucial anti-cancer mechanisms, and require the activity of potent tumor suppressor proteins, such as p53, which are frequently mutated in both heritable and spontaneous cancers (3). Caretakers are also considered to be tumor suppressors because genomic instability drives cancer progression (4) and some heritable forms of cancer are due to mutations in DNA repair genes (5). Unlike gatekeepers, defects in caretakers are not commonly seen in spontaneous tumors. Thus, gatekeepers appear to be more important than caretakers for suppressing spontaneous tumors. Nonhomologous end joining (NHEJ) repairs DNA double-strand breaks (DSBs) by joining ends without using a homologous template strand, and has been described as a caretaker (6,7). Known components of mammalian NHEJ include Ku70, Ku80, DNA-PKCS, Artemis, Xrcc4, DNA ligase IV and Xrcc4-like factor. Ku70 and Ku80 form a heterodimer (termed Ku) that complexes with the Epoxomicin 460 kD DNA-PKCSto form a holoenzyme referred to as DNA-PK (DNA dependent-protein kinase). Artemis and DNA-PKCSform a complex that opens hairpins and processes overhangs. These DNA ends are then ligated by Xrcc4-DNA ligase IV in a complex with Xrcc4-like factor. By rapidly processing and ligating broken DNA, NHEJ suppresses general genomic instability. Ku80-deletion causes an increase in the incidence of DNA DSBs (8) and chromosomal aberrations (9) and also increases the incidence of cancer in mice that carry mutations in additional genes likep53(10,11). Based on these observations, Ku80 has been termed a tumor suppressor in the Epoxomicin caretaker category (6,7). But this interpretation should be viewed with caution since p53-defective mice are rare or nonexistent in nature; thus, Ku80 may not have been selected for a caretaker function. Interestingly,ku80/mice have a reduced, rather than an increased, cancer incidence, although they age prematurely and have a shortened life span (9,12,13). We proposed that the low cancer incidence inku80/mice is due to the Ku80 deletion, and is not an indirect consequence of the shortened life span. We further hypothesized thatku80/cells exhibit persistent gatekeeper responses, owing to inefficiently repaired DNA breaks, and that these responses ultimately impede oncogenesis. To test this hypothesis, we analyzedku80/mice in cancer-prone backgrounds that have either defective or intact gatekeeper responses, predicting that Ku80 deletion will exacerbate oncogenesis in gatekeeper-defective mice but ameliorate oncogenesis in gatekeeper-intact mice. In support of this idea, we previously showed that Ku80 deletion enhances p53-dependent cellular senescence in cultured fibroblasts (10) and exacerbates the development of pro-B cell lymphomas (10) and medulloblastomas (11) in p53-deficient mice. Thus, p53 impacts the Ku80-mutant phenotype to enhance cellular senescence and suppress at least two forms of cancer. However, neither pro-B cell lymphoma nor medulloblastoma are common spontaneous tumors in mice, leaving open the question of whether gatekeeper responses are indeed responsible for the low levels of spontaneous cancer inku80/mice. Here, we evaluate the impact of Ku80 deletion on spontaneous oncogenesis, and investigate the possibility that Ku80 deficiency induces p53-mediated DNA damage responses to poorly repaired DNA lesions. To study spontaneous oncogenesis, we analyzedAPCMINmice deleted for Ku80 because they develop spontaneous intestinal tumors at high incidence within the life span ofku80/mice. Importantly,APCMINmice retain functional DNA damage checkpoints (14). We show that Ku80 deletion significantly reduces tumor burden inAPCMINmice..
