ZNF451 promotes DNA damage repair by recruiting 53BP1/MDC1 in A549 and HeLa cells / 中华放射医学与防护杂志

Objective:To investigate the role of SUMO E3 ligase ZNF451 in DNA damage repair and explore the underlying mechanism in non-small cell lung cancer A549 cells and cervical cancer HeLa cells.Methods:A549 cells and HeLa cells were irradiated with γ-ray irradiation or treated with etoposide. Cell proliferation viability was detected by the cell counting kit-8 assay. Protein expression was detected by Western blot assay. DNA damage repair level was detected by DR-GFP plasmid system, and the spatial positioning was detected by immunofluorescence.Results:Etoposide decreased the expression level of ZNF451 in a dose- and time- dependent manner. After treatment with 30, 50, 80 μmol/L etoposide, the cell viability were reduced after the knockdown of ZNF451 in A549 and HeLa cells(A549: t = 27.62, 25.61, 5.32, P<0.01; HeLa: t = 30.77, 21.28, 4.18, P<0.01). Furthermore, ZNF451 was recruited at DNA damage sites. A co-localization and endogenous interaction were found between ZNF451 and γ-H2AX after the treatment of irradiation or etoposide. Moreover, the expression level of γ-H2AX was significantly increased after treatment with 30, 50, 80 μmol/L etoposide(A549: t = 6.12, 10.67, 4.68, P<0.01; HeLa: t = 7.94, 9.81, 15.12, P<0.01)and the repair efficiency of NHEJ was reduced in ZNF451 knockdown cells( t = 18.60, P<0.05). Finally, the immunofluorescence assay showed that ZNF451 was co-localizated with 53BP1 and MDC1 after irradiation or etoposide treatment. Conclusions:Knockdown of ZNF451 inhibits cell proliferation and increases the level of DNA damage in A549 and HeLa cells. ZNF451 was recruited to DNA damage sites after DSBs and participated in NHEJ repair by co-localizing with DNA damage repair factor 53BP1/MDC1.

Regulation of DNA double-strand break repair pathway choice: a new focus on 53BP1 / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Maintenance of cellular homeostasis and genome integrity is a critical responsibility of DNA double-strand break (DSB) signaling. P53-binding protein 1 (53BP1) plays a critical role in coordinating the DSB repair pathway choice and promotes the non-homologous end-joining (NHEJ)-mediated DSB repair pathway that rejoins DSB ends. New insights have been gained into a basic molecular mechanism that is involved in 53BP1 recruitment to the DNA lesion and how 53BP1 then recruits the DNA break-responsive effectors that promote NHEJ-mediated DSB repair while inhibiting homologous recombination (HR) signaling. This review focuses on the up- and downstream pathways of 53BP1 and how 53BP1 promotes NHEJ-mediated DSB repair, which in turn promotes the sensitivity of poly(ADP-ribose) polymerase inhibitor (PARPi) in BRCA1-deficient cancers and consequently provides an avenue for improving cancer therapy strategies.

Effect of silencing 53BP1 gene on radiosensitivity to esophageal cancer ECA109 cell xenograft in nude mice / 中国癌症杂志

Background and purpose:p53 binding protein (53BP1) expresses in many normal and tumor cells. In vitroexperiments have conifrmed that inhibition of the protein expression of 53BP1 can effectively eliminate cycle arrest of tumor cells, and increase the radiosensitivity after irradiation. However, thein vivo experiment has not been re-ported. This study aimed to explore the effect of silencing 53BP1 gene on the growth and radiosensitivity to esophageal cancer cell ECA109 xenograft in nude mice.Methods:Forty-eight male BALB/c/nu nude mice were randomly divided into 6 groups: ECA109, ECA109/R, ECA109/N, ECA109/NR, ECA109/B and ECA109/BR. Three kinds of prepared cells (ECA109, ECA109/N and ECA109/B) were subcutaneously injected into the paw pads of mice (2×106/100 μL per mouse). The nude mice in ECA109/R, ECA109/NR, and ECA109/BR groups were irradiated with 15 Gy. Tumor growth was monitored every other day on the 6th day after injection. Tumor volume was measured with calipers. Theexpression levels of CHK1, CHK2 and phosphorylated CHK2-T68 protein were examined in different groups by West-ern blot. Apoptotic cell and cell cycle distribution were detected by lfow cytometry assay.Results:Visible tumors were detectable by day 7 after implantation, and the tumor volumes showed no signiifcant differences among all the groups (F=0.67, P=0.69). After irradiation with 15 Gy, tumor volumes in ECA109/BR group were smaller than those in other groups (P=0.03); the growth inhibition rate increased, but the relative growth rate decreased signiifcantly (P=0.01). The q value which relfected the radiosensitizing effect in ECA109/BR group was 1.45. The expressions of CHK1 and CHK2 at protein level in ECA109/BR group were not inlfuenced (P=0.71). However, the level of phosphorylated CHK2-T68 expression decreased signiifcantly after irradiation with 15 Gy (P=0.03). Cell cycle distribution and apoptosis were not signiifcantly different among all the groups (P=0.45).Conclusion:Silencing 53BP1 gene expression could inhibit the growth of esophageal cancer cell xenograft and increase the radiosensitivity to tumors in the nude mice.

DNA Double Strand Breaks in Gastric Epithelium with Helicobacter pylori Infection / 대한소화기학회지

BACKGROUND/AIMS: DNA double strand breaks (DSB) is one of the critical types of DNA damage. If unrepaired, DSB is accumulated in the nucleus of cells, the cells become apoptotic or transform to tumor by way of genomic instability. Some of malignant cancers and its premalignant lesions were proven to have DSB in their nuclei. There was no report that Helicobacter pylori (H. pylori), the gastric carcinogen, induce DNA DSB in gastric epithelium in vivo. The aim of this study was to investigate whether H. pylori induce DSB in the gastric epithelial cells of chronic gastritis. METHODS: Immunohistochemical stains were performed for the DSB markers, phospho-53BP1 and gammaH2AX, in the gastric epithelium derived from 44 peptic ulcer disease patients before and after H. pylori eradication. DNA fragmentation assay was performed in the cell line to investigate the DNA damage by H. pylori infection. RESULTS: The mean expression score of gammaH2AX was significantly higher in the H. pylori infected gastric epithelium as compared to the H. pylori eradicated gastric epithelium (8.8+/-5.5 vs. 6.2+/-5.3 respectively; p=0.008). The expression score of phospho-53BP1 between before and after eradication of H. pylori was not statistically different, but tended to be higher in H. pylori infection. DNA fragmentation was developed significantly more in the cell lines after infection with H. pylori. CONCLUSIONS: DSB of DNA damage was typical feature of H. pylori infection in the gastric epithelium.

Double Strand Break of DNA in Gastric Adenoma and Adenocarcinoma / 대한소화기학회지

BACKGROUND/AIMS: DNA double strand break (DSB) is one of the critical types of DNA damage. When unrepaired DSB is accumulated in the nucleus of the cells having mutations in such genes as p53, it will lead to chromosomal instability and further more to mutation of tumor-activating genes resulting in tumorogenesis. Some of malignant cancers and its premalignant lesions were proven to have DSB in their nuclei. The aim of this study was to define the differences in expression of 53BP1 and gamma-H2AX, the markers of DSB, among normal, gastric adenoma, and gastric adenocarcinoma tissues. METHODS: Tissue microarray was made with the tissues taken from 121 patients who underwent gastrectomy for gastric adenocarcinoma, and 51 patients who underwent endoscopic mucosal resection for gastric adenoma. Immunochemical stain was performed for the marker of DSB, 53BP1 and gamma-H2AX in the tissue microarray. The normal tissues were collected from histologically confirmed tissues with no cellular atypia obtained from the patients with gastric adenocarcinoma. RESULTS: In gastric carcinoma cells, 53BP1 and gamma-H2AX were highly expressed as compared to normal epithelial cells and gastric adenoma (p<0.01). There were no differences in the expression of 53BP1 and gamma-H2AX between normal epithelium and gastric adenoma. The expression of 53BP1 in the adenoma with grade II and III atypism was more elevated than in those with grade I atypism. The expression of 53BP1 and gamma-H2AX were not significantly different according to the clinicopathologic parameters in the patients with gastric adenocarcinoma. CONCLUSIONS: The DSB in DNA seems to be associated with the development of gastric adenocarcinoma, but does not affect the premalignant adenoma cells.

Ser1778 of 53BP1 Plays a Role in DNA Double-strand Break Repairs

53BP1 is an important genome stability regulator, which protects cells against double-strand breaks. Following DNA damage, 53BP1 is rapidly recruited to sites of DNA breakage, along with other DNA damage response proteins, including gamma-H2AX, MDC1, and BRCA1. The recruitment of 53BP1 requires a tandem Tudor fold which associates with methylated histones H3 and H4. It has already been determined that the majority of DNA damage response proteins are phosphorylated by ATM and/or ATR after DNA damage, and then recruited to the break sites. 53BP1 is also phosphorylated at several sites, like other proteins after DNA damage, but this phosphorylation is not critically relevant to recruitment or repair processes. In this study, we evaluated the functions of phosphor-53BP1 and the role of the BRCT domain of 53BP1 in DNA repair. From our data, we were able to detect differences in the phosphorylation patterns in Ser25 and Ser1778 of 53BP1 after neocarzinostatin-induced DNA damage. Furthermore, the foci formation patterns in both phosphorylation sites of 53BP1 also evidenced sizeable differences following DNA damage. From our results, we concluded that each phosphoryaltion site of 53BP1 performs different roles, and Ser1778 is more important than Ser25 in the process of DNA repair.