Regulatory effects of miR-188-5p/XRCC5 on the progression of natural killer/T-cell lymphoma.

PURPOSE: Natural killer/T cell lymphoma (NKTCL) is a malignant condition. The molecular pathological mechanism of NKTCL has not been well studied. In this article we tried to study the role of microRNA-188-5p (miR-188-5p) in NKTCL. METHODS: The expression level of miR-188-5p and XRCC5 was examined by quantitative real-time polymerase chain reaction (qRT-PCR). Cell counting kit-8 (CCK-8) assay and colony formation assay were used to assess the ability of cell proliferation. Dual luciferase reporter assay was used to examine the down-stream target of miR-188-5p. Western blotting was utilized to determine XRCC5 expression level. RESULTS: miR-188-5p was down-regulated in NKTCL. High expression of miR-188-5p accelerated cell proliferation. XRCC5 was one of the down-stream targets. Our data indicated that miR-188-5p suppressed NKTCL progression via regulating XRCC5 expression. CONCLUSIONS: This research elucidated that miR-188-5p suppressed tumor progression in NKTCL by regulating XRCC5. Our data may provide more evidence in looking for novel therapeutic targets.

Ku70 and Ku80 participate in LPS-induced pro-inflammatory cytokines production in human macrophages and monocytes.

In human macrophages and monocytes, lipopolysaccharide (LPS) induces nuclear factor kappa B (NFκB) activation and pro-inflammatory cytokines production. We tested the possible involvement of Ku70 and Ku80 in the process. In THP-1 macrophages and primary human peripheral blood mononuclear cells (PBMCs), shRNA-induced double knockdown of Ku70 and Ku80 potently inhibited LPS-induced production of pro-inflammatory cytokines (TNF-α, IL-1ß and IL-6). Additionally, we developed CRISPR/Cas-9 gene-editing methods to knockout both Ku70 and Ku80 in THP-1 cells and PBMCs. Double knockout (DKO) largely inhibited LPS-induced pro-inflammatory cytokines production. Conversely, in THP-1 cells exogenous overexpression of both Ku70 and Ku80 enhanced the pro-inflammatory cytokines production by LPS. Ku70 and Ku80 co-immunoprecipitated with p65-p52 NFκB complex in the nuclei of LPS-treated THP-1 cells. Significantly, LPS-induced NFκB activation was inhibited by Ku70 plus Ku80 double knockdown or DKO. It was however enhanced with Ku70 and Ku80 overexpression. Together, Ku70 and Ku80 promote LPS-induced NFκB activation and pro-inflammatory response in THP-1 cells and human PBMCs.

Canonical DNA non-homologous end-joining; capacity versus fidelity.

The significance of canonical DNA non-homologous end-joining (c-NHEJ) for DNA double strand break (DSB) repair has increased from lower organisms to higher eukaryotes, and plays the predominant role in human cells. Ku, the c-NHEJ end-binding component, binds DSBs with high efficiency enabling c-NHEJ to be the first choice DSB repair pathway, although alternative pathways can ensue after regulated steps to remove Ku. Indeed, radiation-induced DSBs are repaired rapidly in human cells. However, an important question is the fidelity with which radiation-induced DSBs are repaired, which is essential for assessing any harmful impacts caused by radiation exposure. Indeed, is compromised fidelity a price we pay for high capacity repair. Two subpathways of c-NHEJ have been revealed; a fast process that does not require nucleases or significant chromatin changes and a slower process that necessitates resection factors, and potentially more significant chromatin changes at the DSB. Recent studies have also shown that DSBs within transcriptionally active regions are repaired by specialised mechanisms, and the response at such DSBs encompasses a process of transcriptional arrest. Here, we consider the limitations of c-NHEJ that might result in DSB misrepair. We consider the common IR-induced misrepair events and discuss how they might arise via the distinct subpathways of c-NHEJ.

XLF and APLF bind Ku80 at two remote sites to ensure DNA repair by non-homologous end joining.

The Ku70-Ku80 (Ku) heterodimer binds rapidly and tightly to the ends of DNA double-strand breaks and recruits factors of the non-homologous end-joining (NHEJ) repair pathway through molecular interactions that remain unclear. We have determined crystal structures of the Ku-binding motifs (KBM) of the NHEJ proteins APLF (A-KBM) and XLF (X-KBM) bound to a Ku-DNA complex. The two KBM motifs bind remote sites of the Ku80 α/ß domain. The X-KBM occupies an internal pocket formed by an unprecedented large outward rotation of the Ku80 α/ß domain. We observe independent recruitment of the APLF-interacting protein XRCC4 and of XLF to laser-irradiated sites via binding of A- and X-KBMs, respectively, to Ku80. Finally, we show that mutation of the X-KBM and A-KBM binding sites in Ku80 compromises both the efficiency and accuracy of end joining and cellular radiosensitivity. A- and X-KBMs may represent two initial anchor points to build the intricate interaction network required for NHEJ.

Role of Ku70 in the apoptosis of inflamed dental pulp stem cells.

AIM: The study aimed to investigate the effects of DNA repair proteins on cell apoptosis in human DPSCs during inflammation. METHODS: Lipopolysaccharide (LPS) was used to stimulate inflammation in dental pulp in vivo and in vitro. We identified the activation of DSB response and DNA repair proteins in inflamed pulp tissue and in LPS-treated human DPSCs. Then we transfected the cells with Ku70 (a key protein involved in NHEJ) siRNA and detected the expression changes of γ-H2A.X, DNA repair proteins and cell apoptosis. RESULTS: Immunohistochemical staining showed that at 4 and 6 days of pulpitis the expression of Ku70 and γ-H2A.X significantly increased. The levels of γ-H2A.X, Ku70, Xrcc4, and Rad51 increased considerably in the LPS-treated DPSCs. Furthermore, decreased expression of Ku70 could increase the number of γ-H2A.X foci, apoptotic cells and reduce cell viability in DPSCs. CONCLUSIONS: The results indicate that NHEJ pathway was the main mechanism involved in DNA damage response induced by repeated LPS stimulation in DPSCs. Meanwhile, the findings suggested that Ku70 serves importantly in the apoptosis of DPSCs in the inflammatory environment.

Repeat-mediated deletions can be induced by a chromosomal break far from a repeat, but multiple pathways suppress such rearrangements.

Chromosomal deletion rearrangements mediated by repetitive elements often involve repeats separated by several kilobases and sequences that are divergent. While such rearrangements are likely induced by DNA double-strand breaks (DSBs), it has been unclear how the proximity of DSBs relative to repeat sequences affects the frequency of such events. We generated a reporter assay in mouse cells for a deletion rearrangement involving repeats separated by 0.4 Mb. We induced this repeat-mediated deletion (RMD) rearrangement with two DSBs: the 5' DSB that is just downstream from the first repeat and the 3' DSB that is varying distances upstream of the second repeat. Strikingly, we found that increasing the 3' DSB/repeat distance from 3.3 kb to 28.4 kb causes only a modest decrease in rearrangement frequency. We also found that RMDs are suppressed by KU70 and RAD51 and promoted by RAD52, CtIP, and BRCA1. In addition, we found that 1%-3% sequence divergence substantially suppresses these rearrangements in a manner dependent on the mismatch repair factor MSH2, which is dominant over the suppressive role of KU70. We suggest that a DSB far from a repeat can stimulate repeat-mediated rearrangements, but multiple pathways suppress these events.

PIDD mediates the association of DNA-PKcs and ATR at stalled replication forks to facilitate the ATR signaling pathway.

The DNA-dependent protein kinase (DNA-PK), consisting of the DNA binding Ku70/80 heterodimer and the catalytic subunit DNA-PKcs, has been well characterized in the non-homologous end-joining mechanism for DNA double strand break (DSB) repair and radiation resistance. Besides playing a role in DSB repair, DNA-PKcs is required for the cellular response to replication stress and participates in the ATR-Chk1 signaling pathway. However, the mechanism through which DNA-PKcs is recruited to stalled replication forks is still unclear. Here, we report that the apoptosis mediator p53-induced protein with a death domain (PIDD) is required to promote DNA-PKcs activity in response to replication stress. PIDD is known to interact with PCNA upon UV-induced replication stress. Our results demonstrate that PIDD is required to recruit DNA-PKcs to stalled replication forks through direct binding to DNA-PKcs at the N' terminal region. Disruption of the interaction between DNA-PKcs and PIDD not only compromises the ATR association and regulation of DNA-PKcs, but also the ATR signaling pathway, intra-S-phase checkpoint and cellular resistance to replication stress. Taken together, our results indicate that PIDD, but not the Ku heterodimer, mediates the DNA-PKcs activity at stalled replication forks and facilitates the ATR signaling pathway in the cellular response to replication stress.

Inhibiting DNA-PKcs in a non-homologous end-joining pathway in response to DNA double-strand breaks.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a distinct factor in the non-homologous end-joining (NHEJ) pathway involved in DNA double-strand break (DSB) repair. We examined the crosstalk between key proteins in the DSB NHEJ repair pathway and cell cycle regulation and found that mouse embryonic fibroblast (MEF) cells deficient in DNA-PKcs or Ku70 were more vulnerable to ionizing radiation (IR) compared with wild-type cells and that DSB repair was delayed. γH2AX was associated with phospho-Ataxia-telangiectasia mutated kinase (Ser1987) and phospho-checkpoint effector kinase 1 (Ser345) foci for the arrest of cell cycle through the G2/M phase. Inhibition of DNA-PKcs prolonged IR-induced G2/M phase arrest because of sequential activation of cell cycle checkpoints. DSBs were introduced, and cell cycle checkpoints were recruited after exposure to IR in nasopharyngeal carcinoma SUNE-1 cells. NU7441 radiosensitized MEF cells and SUNE-1 cells by interfering with DSB repair. Together, these results reveal a mechanism in which coupling of DSB repair with the cell cycle radiosensitizes NHEJ repair-deficient cells, justifying further development of DNA-PK inhibitors in cancer therapy.

Scaffold attachment factor A (SAF-A) and Ku temporally regulate repair of radiation-induced clustered genome lesions.

Ionizing radiation (IR) induces highly cytotoxic double-strand breaks (DSBs) and also clustered oxidized bases in mammalian genomes. Base excision repair (BER) of bi-stranded oxidized bases could generate additional DSBs as repair intermediates in the vicinity of direct DSBs, leading to loss of DNA fragments. This could be avoided if DSB repair via DNA-PK-mediated nonhomologous end joining (NHEJ) precedes BER initiated by NEIL1 and other DNA glycosylases (DGs). Here we show that DNA-PK subunit Ku inhibits DGs via direct interaction. The scaffold attachment factor (SAF)-A, (also called hnRNP-U), phosphorylated at Ser59 by DNA-PK early after IR treatment, is linked to transient release of chromatin-bound NEIL1, thus preventing BER. SAF-A is subsequently dephosphorylated. Ku inhibition of DGs in vitro is relieved by unphosphorylated SAF-A, but not by the phosphomimetic Asp59 mutant. We thus propose that SAF-A, in concert with Ku, temporally regulates base damage repair in irradiated cell genome.