DNA damage response pathways in cancer causation and treatment

Cellular responses to DNA damage impact many aspects of cancer biology. First, damage to cellular DNA causes cancer. We know this from epidemiologic studies, from animal models, and from the observation that many human cancer susceptibility syndromes arise from mutations in genes involved in DNA damage responses. For example, the genes mutated in Fanconi's anemia, ataxia-telangiectasia, xeroderma pigmentosum, Li–Fraumeni syndrome, hereditary breast and ovarian cancers, and hereditary non-polyposis colon cancer are all involved in DNA damage responses. Second, DNA damage is used to cure cancer. The majority of the therapeutic modalities that we currently use to treat malignancies target the DNA, including radiation therapy and many chemotherapeutic agents. Third, DNA damage is responsible for the majority of the side effects of therapy. Bone marrow suppression, GI toxicities, and hair loss are all attributable to DNA damage-induced cellular apoptosis of proliferating progenitor cells in these tissues. Thus, DNA damage causes the disease, is used to treat the disease, and is responsible for the toxicity of therapies for the disease. Significant progress has been made in recent years in elucidating the molecular controls of cellular responses to DNA damage in mammalian cells. These insights now provide us with approaches to attempt to manipulate these responses for patient benefit, such as enhanced tumor cell kill with therapy, protection of normal tissues from toxic effects of therapy, and even prevention of cancer development. Many of the insights that we have gained into the mechanisms involved in cellular DNA damage response pathways have come from studies of human cancer susceptibility syndromes that are altered in DNA damage responses. One of these disorders, ataxia-telangiectasia (A-T), is characterized by multiple physiologic abnormalities, including neurodegeneration, immunologic abnormalities, cancer predisposition, sterility, and metabolic abnormalities. The gene mutated in this disorder, Atm, is a protein kinase that is activated by the introduction of DNA double-strand breaks in cells. Atm activity is required for cell cycle arrests induced by ionizing irradiation (IR) in G1, S, and G2 phases of the cell cycle. Several targets of the Atm kinase have been identified that participate in these IR-induced cell cycle arrests. For example, phosphorylation of p53, mdm2, and Chk2 participate in the G1 checkpoint; Nbs1, Brca1, FancD2, and Smc1 participate in the transient IR-induced S-phase arrest; and Brca1 and hRad17 have been implicated in the G2/M checkpoint. Although Atm is critical for cellular responses to IR, related kinases, such as Atr, appear to be important for responses to other cellular stresses [1]. Some substrates appear to be shared by the two kinases, with the major difference being which stimulus is present and which kinase is used to initiate the signaling pathway. Characterization of these Atm substrates permitted us to manipulate these proteins in cell lines and to selectively abrogate single or multiple checkpoints. Using this approach, we demonstrated that abrogation of checkpoints does not by itself result in radiosensitivity. Although this has been known for several years in regards to the S-phase checkpoint, it was a surprising finding that abrogation of the G2/M checkpoint did not cause radiosensitivity. This observation suggested that some other function of Atm, other than checkpoint control, was important for cellular survival following ionizing irradiation. In characterizing targets of the Atm kinase, the only substrate whose phosphorylation seems to impact on radiosensitivity is Smc1 [2]. We previously demonstrated that the phosphorylation of Smc1 by ATM required the presence of both Nbs1 and Brca1 proteins. We recently found that this dependence results from the role that these two proteins play in recruiting both Smc1 protein and activated Atm to the sites of DNA breaks. We generated mice in which the two Atm phosphorylation sites in the Smc1 protein are mutated; cells from these mice demonstrate normal ATM activation, normal phosphorylation of both Nbs1 and Brca1 after IR, and normal migration of these proteins to DNA breaks [3]. Despite these normal activities of Atm, Nbs1 and Brca1, these cells exhibit a defective S-phase checkpoint, radiosensitivity, and increased chromosomal breakage after IR similar to that seen in cells lacking Atm. These results suggest that the phosphorylation of Smc1 is the critical target of this signaling pathway for these endpoints, and that the reason why cells lacking Nbs1 and Brca1 are radiosensitive and exhibit chromosomal breakage is due to a failure to recruit Smc1 to the sites of DNA breaks where it gets phosphorylated by previously activated Atm. Recent studies also elucidated the mechanism by which DNA damage activates the Atm kinase and initiates these critical cellular signaling pathways [4]. Atm normally exists as an inactive homodimer bound to nuclear chromatin in unperturbed cells, and introduction of DNA damage induces intermolecular autophosphorylation on serine 1981 in both Atm molecules. This phosphorylation causes a dissociation of the Atm molecules and frees it up to now circulate around the cell and phosphorylate the substrates that regulate cell cycle progression and DNA repair processes. This regulation of Atm activity in the cell represents a novel mechanism of protein kinase regulation and appears to result from alterations in higher order chromatin structure rather than direct binding of Atm to DNA strand breaks. Although Nbs1 and Brca1 are not required for the initial activation of Atm after IR, these two proteins are required for the migration of activated Atm to the sites of DNA breaks. It is this process of recruitment of activated Atm along with Smc1 recruitment to the DNA breaks that leads to Smc1 phosphorylation by Atm and presumably initiation of some repair process(es) that reduce chromosomal breakage and enhance cell survival.