Congenital bone marrow failure in DNA-PKcs mutant mice associated with deficiencies in DNA repair

The nonhomologous end-joining (NHEJ) pathway is essential for radioresistance and lymphocyte-specific V(D)J (variable [diversity] joining) recombination. Defects in NHEJ also impair hematopoietic stem cell (HSC) activity with age but do not affect the initial establishment of HSC reserves. In this paper, we report that, in contrast to deoxyribonucleic acid (DNA)–dependent protein kinase catalytic subunit (DNA-PKcs)–null mice, knockin mice with the DNA-PKcs(3A/3A) allele, which codes for three alanine substitutions at the mouse Thr2605 phosphorylation cluster, die prematurely because of congenital bone marrow failure. Impaired proliferation of DNA-PKcs(3A/3A) HSCs is caused by excessive DNA damage and p53-dependent apoptosis. In addition, increased apoptosis in the intestinal crypt and epidermal hyperpigmentation indicate the presence of elevated genotoxic stress and p53 activation. Analysis of embryonic fibroblasts further reveals that DNA-PKcs(3A/3A) cells are hypersensitive to DNA cross-linking agents and are defective in both homologous recombination and the Fanconi anemia DNA damage response pathways. We conclude that phosphorylation of DNA-PKcs is essential for the normal activation of multiple DNA repair pathways, which in turn is critical for the maintenance of diverse populations of tissue stem cells in mice

DNA Repair Processes and Checkpoint Pathways in Human Cells Exposed to Heavy Ion Beams

The DNA double-strand break (DSB) is the most deleterious of the ionizing radiation- induced DNA damages. Two major repair pathways for DSBs have been well studied, nonhomologous end-joining and homologous recombination. It is known that high linear energy transfer radiation, such as heavy ion beams, induces complex DSBs with clustered damages at the end and that, as a result, the efficiency of nonhomologous end- joining in repairing these DSBs is diminished. We have shown that more than 80% of complex DSBs in S/G2 human cells are subjected to DNA end resection, an early step in homologous recombination to generate single-strand DNA. Furthermore, recent work, including ours, revealed that a subpopulation of human G1 cells exhibit resection activity following ionizing radiation, which is dependent on CtIP, as in other cell cycle phases, and also dependent on the complexity of the DSB. Collectively, this recent progress indicates that the complexity of the DSB structure drastically enhances end resection, with CtIP being a significant factor required for complex DSB repair throughout the cell cycle. We further revealed that the ATR pathway, which is activated by end resection, plays a pivotal role in regulating early G2/M arrest in ATM-deficient cells exposed to high linear energy transfer ion beams. This suggests that the complexity of the DSB also influences the choice of the signaling pathway via the enhanced resection. Additionally, we discuss a possibility that CtIP has an additional function (or functions) after the initiation of resection. In conclusion, new findings and insight are pivotal to allow innovative progress in heavy ion-particle therapy by shedding light on the whole response at the molecular level in cells exposed to heavy ion beams

Congenital bone marrow failure in DNA-PKcs mutant mice associated with deficiencies in DNA repair

The nonhomologous end-joining (NHEJ) pathway is essential for radioresistance and lymphocyte- specific V(D)J (variable [diversity] joining) recombi- nation. Defects in NHEJ also impair hematopoietic stem cell (HSC) activity with age but do not affect the initial establishment of HSC reserves. In this paper, we report that, in contrast to deoxyribonucleic acid (DNA)–dependent protein kinase catalytic subunit (DNA-PKcs)–null mice, knockin mice with the DNA-PKcs3A/3A allele, which codes for three alanine substitutions at the mouse Thr2605 phos- phorylation cluster, die prematurely because of congenital bone marrow failure. Impaired proliferation of DNA- PKcs3A/3A HSCs is caused by excessive DNA damage and p53-dependent apoptosis. In addition, increased apopto- sis in the intestinal crypt and epidermal hyperpigmenta- tion indicate the presence of elevated genotoxic stress and p53 activation. Analysis of embryonic fibroblasts further reveals that DNA-PKcs3A/3A cells are hypersensitive to DNA cross-linking agents and are defective in both homologous recombination and the Fanconi anemia DNA damage response pathways. We conclude that phosphory- lation of DNA-PKcs is essential for the normal activation of multiple DNA repair pathways, which in turn is critical for the maintenance of diverse populations of tissue stem cells in mice

Involvement of DNA-dependent protein kinase in normal cell cycle progression through mitosis

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) plays an important role in DNA double-strand break (DSB) repair as the underlying mechanism of the non- homologous end joining pathway. When DSBs occur, DNA- PKcs is rapidly phosphorylated at both the Thr-2609 and Ser- 2056 residues, and such phosphorylations are critical for DSB repair. In this study we report that, in addition to responding to DSBs, DNA-PKcs is activated and phosphorylated in normal cell cycle progression through mitosis. Mitotic induction of DNA- PKcs phosphorylation is closely associated with the spindle apparatus at centrosomes and kinetochores. Furthermore, depletion of DNA-PKcs protein levels or inhibition of DNA- PKcs kinase activity results in the delay of mitotic transition because of chromosome misalignment. These results demon- strate for the first time that DNA-PKcs, in addition to its role in DSB repair, is a critical regulator of mitosis and could modulate microtubule dynamics in chromosome segregation

Cancer stem-like cells from a glioblastoma cell line is resistant to X-rays and carbon-ion beams

Radiotherapy is one of the established treatments for solid tumors. Investigation of tumor radioresistance is important for future tumor radiotherapy. Biological bases on the resistance have not been fully understood. Recent studies have demonstrated that there is a small fraction in solid tumor which is highly resistant to ionizing radiation and the radioresistant tumor cells often expressed some stem cell markers. Resistance of tumors to both radiation and chemotherapeutic agents can be attributed to the features of the cancer stem cells. In the present study, we show that a human glioblastoma cell line A172 transiently becomes to cancer stem cell-like when cultured with non-serum media which is supplemented with growth factors. The treated cell was found significantly resistant to X-rays and heavy ion particles (carbon) compared with the A172 cells cultured with the normal media. Phosphorylation of histon H2Ax was induced in both forms, however, recovered earlier in the stem cell-like population than in the parental cells. Our result is consistent with the hypothesis that cancer stemness causes radioresistance through the efficient activity of DNA double-strand break repair.第70回日本癌学会学術総

ATR-Dependent Phosphorylation of DNA-Dependent Protein Kinase Catalytic Subunit in Response to UV-Induced Replication Stress

Phosphorylation of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) upon ionizing radiation (IR) is essential for cellular radioresistance and nonhomologous-end-joining-mediated DNA double-strand break repair. In addition to IR induction, we have previously shown that DNA-PKcs phosphorylation is increased upon camptothecin treatment, which induces replication stress and replication-associated double-strand breaks. To clarify the involvement of DNA-PKcs in this process, we analyzed DNA-PKcs phosphorylation in response to UV irradiation, which causes replication stress and activates ATR (ATM-Rad3-related)/ATM (ataxia-telangiectasia mutated) kinases in a replication-dependent manner. Upon UV irradiation, we observed a rapid DNA-PKcs phosphorylation at T2609 and T2647, but not at S2056, distinct from that induced by IR. UV-induced DNA-PKcs phosphorylation occurs specifically only in replicating cells and is dependent on ATR kinase. Inhibition of ATR activity via caffeine, a dominant-negative kinase-dead mutant, or RNA interference led to the attenuation of UV-induced DNA-PKcs phosphorylation. Furthermore, DNA-PKcs associates with ATR in vivo and is phosphorylated by ATR in vitro, suggesting that DNA-PKcs could be the direct downstream target of ATR. Taken together, these results strongly suggest that DNA-PKcs is required for the cellular response to replication stress and might play an important role in the repair of stalled replication forks