Discordance between phosphorylation and recruitment of 53BP1 in response to DNA double-strand breaks

During the DNA damage response (DDR), chromatin modifications contribute to localization of 53BP1 to sites of DNA double-strand breaks (DSBs). 53BP1 is phosphorylated during the DDR, but it is unclear whether phosphorylation is directly coupled to chromatin binding. In this study, we used human diploid fibroblasts and HCT116 tumor cells to study 53BP1 phosphorylation at Serine-25 and Serine-1778 during endogenous and exogenous DSBs (DNA replication and whole-cell or sub-nuclear microbeam irradiation, respectively). In non-stressed conditions, endogenous DSBs in S-phase cells led to accumulation of 53BP1 and γH2AX into discrete nuclear foci. Only the frank collapse of DNA replication forks following hydroxyurea treatment initiated 53BP1^Ser25 and 53BP1^Ser1778 phosphorylation. In response to exogenous DSBs, 53BP1^Ser25 and 53BP1^Ser1778 phosphoforms localized to sites of initial DSBs in a cell cycle-independent manner. 53BP1 phosphoforms also localized to late residual foci and associated with PML-NBs during IR-induced senescence. Using isogenic cell lines and small-molecule inhibitors, we observed that DDR-induced 53BP1 phosphorylation was dependent on ATM and DNA-PKcs kinase activity but independent of MRE11 sensing or RNF168 chromatin remodeling. However, loss of RNF168 blocked recruitment of phosphorylated 53BP1 to sites of DNA damage. Our results uncouple 53BP1 phosphorylation from DSB localization and support parallel pathways for 53BP1 biology during the DDR. As relative 53BP1 expression may be a biomarker of DNA repair capacity in solid tumors, the tracking of 53BP1 phosphoforms in situ may give unique information regarding different cancer phenotypes or response to cancer treatment.     

DNA repair mechanisms in dividing and non-dividing cells

DNA damage created by endogenous or exogenous genotoxic agents can exist in multiple forms, and if allowed to persist, can promote genome instability and directly lead to various human diseases, particularly cancer, neurological abnormalities, immunodeficiency and premature aging. To avoid such deleterious outcomes, cells have evolved an array of DNA repair pathways, which carry out what is typically a multiple-step process to resolve specific DNA lesions and maintain genome integrity. To fully appreciate the biological contributions of the different DNA repair systems, one must keep in mind the cellular context within which they operate. For example, the human body is composed of non-dividing and dividing cell types, including, in the brain, neurons and glial cells. We describe herein the molecular mechanisms of the different DNA repair pathways, and review their roles in non-dividing and dividing cells, with an eye toward how these pathways may regulate the development of neurological disease.     

DNA-PKcs, but not TLR9, is required for activation of Akt by CpG-DNA

CpG-DNA and its related synthetic CpG oligodeoxynucleotides (CpG-ODNs) play an important role in immune cell survival. It has been suggested that Akt is one of the CpG-DNA-responsive serine/threonine kinases; however, the target protein of CpG-DNA that leads to Akt activation has not been elucidated. Here, we report that ex vivo stimulation of bone marrow-derived macrophages (BMDMs) from mice lacking the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) results in defective phosphorylation and activation of Akt by CpG-DNA. Unexpectedly, loss of the Toll-like receptor 9 has a minimal effect on Akt activation in response to CpG-DNA. Further in vitro analysis using purified DNA-PK and recombinant Akt proteins reveals that DNA-PK directly induces phosphorylation and activation of Akt. In addition, in BMDMs, DNA-PKcs associates with Akt upon CpG-DNA stimulation and triggers transient nuclear translocation of Akt. Thus, our findings establish a novel role for DNA-PKcs in CpG-DNA signaling and define a CpG-DNA/DNA-PKcs/Akt pathway.     

The role of nitric oxide in cancer

Nitric oxide (NO) is a pleiotropic regulator, critical to numerous biological processes, including va-sodilatation, neurotransmission and macrophage-mediated immunity. The family of nitric oxide synthases (NOS) comprises inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS). Interestingly, various studies have shown that all three isoforms can be involved in promoting or inhibiting the etiology of cancer. NOS activity has been detected in tumour cells of various histogenetic origins and has been associated with tumour grade, proliferation rate and expression of important signaling components associated with cancer development such as the oestrogen receptor. It appears that high levels of NOS expression (for example, generated by activated macrophages) may be cytostatic or cytotoxic for tumor cells, whereas low level activity can have the opposite effect and promote tumour growth. Paradoxically therefore, NO (and related reactive nitrogen species) may have both genotoxic and angiogenic pro     

Ferulic acid (FA) abrogates γ-radiation induced oxidative stress and DNA damage by up-regulating nuclear translocation of Nrf2 and activation of NHEJ pathway

The present study was aimed to evaluate the radioprotective effect of ferulic acid (FA), a naturally occurring plant flavonoid in terms of DNA damage and damage related alterations of repair pathways by gamma radiation. FA was administered at a dose of 50?mg/kg body weight for five consecutive days prior to exposing the swiss albino mice to a single dose of 10?Gy gamma radiation. Ionising radiation induces oxidative damage manifested by decreased expression of Cu, Zn-SOD (SOD stands for super oxide dismutase), Mn-SOD and catalase. Gamma radiation promulgated reactive oxygen species (ROS) mediated DNA damage and modified repair pathways. ROS enhanced nuclear translocation of p53, activated ATM (ataxia telangiectasia-mutated protein), increased expression of GADD45a (growth arrest and DNA-damage-inducible protein) gene and inactivated Non homologous end joining (NHEJ) repair pathway. The comet formation in irradiated mice peripheral blood mononuclear cells (PBMC) reiterated the DNA damage in IR exposed groups. FA pretreatment significantly prevented the comet formation and regulated the nuclear translocation of p53, inhibited ATM activation and expression of GADD45a gene. FA promoted the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and activated NHEJ repair pathway to overcome ROS mediated oxidative stress and DNA damage. Therefore, the current study stated that FA can challenge the oxidative stress by (i) inducing nuclear translocation of Nrf2, (ii) scavenging ROS, and (iii) activating NHEJ DNA repair process.     

DNA双链断裂的非同源末端连接修复

细胞内普遍存在的DNA双链断裂(DSB)可通过同源重组(HR)或非同源末端连接(NHEJ)修复。由于HR仅在存在相同染色体作为模板的时候进行,因此,NHEJ通常为主要的修复方式。在NHEJ中,DSB末端首先由Ku识别,接着由核酸酶、聚合酶在Ku与DNA-PKcs协助下加工,并由连接酶IVXRCC4-XLF连接。NHEJ底物类型多样,末端的修复常包含反复加工的过程,导致修复产物通常无法复原损伤前的序列。虽然无法确保准确修复DNA,NHEJ仍对维持基因组的稳定性具有重要的意义。对NHEJ的研究有助于理解癌症的发生机制并将促进癌症的治疗。     

Knockdown of AMPKα decreases ATM expression and increases radiosensitivity under hypoxia and nutrient starvation in an SV40-transformed human fibroblast cell line,LM217

Background

Presence of unperfused regions containing cells under hypoxia and nutrient starvation contributes to radioresistance in solid human tumors. It is well known that hypoxia causes cellular radioresistance, but little is known about the effects of nutrient starvation on radiosensitivity. We have reported that nutrient starvation induced decrease of mTORC1 activity and decrease of radiosensitivity in an SV40-transformed human fibroblast cell line, LM217, and that nutrient starvation induced increase of mTORC1 activity and increase of radiosensitivity in human liver cancer cell lines, HepG2 and HuH6 (Murata et al., BBRC 2015). Knockdown of mTOR using small interfering RNA (siRNA) for mTOR suppressed radiosensitivity under nutrient starvation alone in HepG2 cells, which suggests that mTORC1 pathway regulates radiosensitivity under nutrient starvation alone. In the present study, effects of hypoxia and nutrient starvation on radiosensitivity were investigated using the same cell lines.

The complexity of DNA double strand break is a crucial factor for activating ATR signaling pathway for G2/M checkpoint regulation regardless of ATM function

DNA double strand break (DSB) repair pathway choice following ionizing radiation (IR) is currently an appealing research topic, which is still largely unclear. Our recent paper indicated that the complexity of DSBs is a critical factor that enhances DNA end resection. It has been well accepted that the RPA-coated single strand DNA produced by resection is a signaling structure for ATR activation. Therefore, taking advantage of high linear energy transfer (LET) radiation to effectively produce complex DSBs, we investigated how the complexity of DSB influences the function of ATR pathway on the G2/M checkpoint regulation. Human skin fibroblast cells with or without ATM were irradiated with X rays or heavy ion particles, and dual-parameter flow cytometry was used to quantitatively assess the mitotic entry at early period post radiation by detecting the cells positive for phosphor histone H3. In ATM-deficient cells, ATR pathway played a pivotal role and functioned in a dose- and LET-dependent way to regulate the early G2/M arrest even as low as 0.2 Gy for heavy ion radiation, which indicated that ATR pathway could be rapidly activated and functioned in an ATM-independent, but DSB complexity-dependent manner following exposure to IR. Furthermore, ATR pathway also functioned more efficiently in ATM-proficient cells to block G2 to M transition at early period of particle radiation exposure. Accordingly, in contrast to ATM inhibitor, ATR inhibitor had a more effective radiosensitizing effect on survival fraction following heavy ion beams as compared with X ray radiation. Taken together, our results reveal that the complexity of DSBs is a crucial factor for the activation of ATR pathway for G2/M checkpoint regulation, and ATM-dependent end resection is not essential for the activation.