Hua Guan

γH2AX foci formation in the absence of DNA damage: Mitotic H2AX phosphorylation is mediated by the DNA-PKcs/CHK2 pathway

Phosphorylated H2AX is considered to be a biomarker for DNA double‐strand breaks (DSB), but recent evidence suggests that γH2AX does not always indicate the presence of DSB. Here we demonstrate the bimodal dynamic of H2AX phosphorylation induced by ionizing radiation, with the second peak appearing when G2/M arrest is induced. An increased level of γH2AX occurred in mitotic cells, and this increase was attenuated by DNA‐PKcs inactivation or Chk2 depletion, but not by ATM inhibition. The phosphorylation‐mimic CHK2‐T68D abrogated the attenuation of mitotic γH2AX induced by DNA‐PKcs inactivation. Thus, the DNA‐PKcs/CHK2 pathway mediates the mitotic phosphorylation of H2AX in the absence of DNA damage.

4E-BP1 participates in maintaining spindle integrity and genomic stability via interacting with PLK1.

The essential function of eIF4E-binding protein 1 (4E-BP1) in translation initiation has been well established; however, the role of 4E-BP1 in normal cell cycle progression is coming to attention. Here, we revealed the role of 4E-BP1 on mitotic regulation and chromosomal DNA dynamics during mitosis. First, we have observed the co-localization of the phosphorylated 4E-BP1 at T37/46 with Polo-like kinase 1 (PLK1) at the centrosomes during. Depression of 4E-BP1 by small interfering RNA in HepG2 or HeLa cells resulted in an increased outcome of polyploidy and aberrant mitosis, including chromosomal DNA misaligned and multi-polar spindles or multiple centrosomes. We observed that 4E-BP1 interacted with PLK1 directly in vitro and in vivo in mitotic cells, and the C-terminal aa 77–118 of 4E-BP1 mediates its interaction with PLK1. PLK1 can phosphorylate 4E-BP1 in vitro. Furthermore, the depletion of 4E-BP1 sensitized HepG2 and HeLa cells to the microtubule disruption agent paclitaxel. These results demonstrate that 4E-BP1, beyond its role in translation regulation, can function as a regulator of mitosis via interacting with PLK1, and possibly plays a role in genomic stability maintaining.

DNA‐PKcs Associates With PLK1 and Is Involved in Proper Chromosome Segregation and Cytokinesis

Accurate mitotic regulation is as important as intrinsic DNA repair for maintaining genomic stability. It is believed that these two cellular mechanisms are interconnected with DNA damage. DNA‐PKcs is a critical component of the non‐homologous end‐joining pathway of DNA double‐stranded break repair, and it was recently discovered to be involved in mitotic processing. However, the underlying mechanism of DNA‐PKcs action in mitotic control is unknown. Here, we demonstrated that depletion of DNA‐PKcs led to the dysregulation of mitotic progression in response to DNA damage, which eventually resulted in multiple failures, including failure to segregate sister chromatids and failure to complete cytokinesis, with daughter cells becoming fused again. The depletion of DNA‐PKcs resulted in a notable failure of cytokinesis, with a high incidence of multinucleated cells. There were also cytoplasmic bridges containing DNA that continuously connected the daughter cells after DNA damage was induced. Phosphorylated DNA‐PKcs (T2609) colocalizes with PLK1 throughout mitosis, including at the centrosomes from prophase to anaphase and at the kinetochores from prometaphase to metaphase, with accumulation at the midbody during cytokinesis. Importantly, DNA‐PKcs was found to associate with PLK1 in the mitotic phase, and the depletion of DNA‐PKcs resulted in the overexpression of PLK1 due to increased protein stability. However, deficiency in DNA‐PKcs attenuated the recruitment of phosphorylated PLK1 to the midbody but not to the kinetochores and centrosomes. Our results demonstrate the functional association of DNA‐PKcs with PLK1, especially in chromosomal segregation and cytokinesis control. J. Cell. Biochem. 115: 1077–1088, 2014.

The involvement of c-Myc in the DNA double-strand break repair via regulating radiation-induced phosphorylation of ATM and DNA-PKcs activity

Deregulation of c-Myc often occurs in various human cancers, which not only contributes to the genesis and progression of cancers but also affects the outcomes of cancer radio- or chemotherapy. In this study, we have investigated the function of c-Myc in the repair of DNA double-strand break (DSB) induced by γ-ray irradiation. A c-Myc-silenced Hela-630 cell line was generated from HeLa cells using RNA interference technology. The DNA DSBs were detected by γ-H2AX foci, neutral comet assay and pulsed-field gel electrophoresis. We found that the capability of DNA DSB repair in Hela-630 cells was significantly reduced, and the repair kinetics of DSB was delayed as compared to the control Hela-NC cells. Silence of c-myc sensitized the cellular sensitivity to ionizing radiation. The phosphorylated c-Myc (Thr58/pSer62) formed the consistent co-localisation foci with γ-H2AX as well as the phosphorylated DNA-PKcs/S2056 in the irradiated cells. Moreover, depression of c-Myc largely attenuated the ionizing radiation-induced phosphorylation of the ataxia telangiectasia mutated (ATM) and decreased the in vitro kinase activity of DNA-PKcs. Taken together, our results demonstrated that c-Myc protein functions in the process of DNA double-strand break repair, at least partially, through affecting the ATM phosphorylation and DNA-PKcs kinase activity. The overexpression of c-Myc in tumours can account for the radioresistance of some tumour cell types.

PIG3 Functions in DNA Damage Response through Regulating DNA-PKcs Homeostasis

The p53-inducible gene 3 (PIG3) recently has been reported to be a new player in DNA damage signaling and response, but the crucial mechanism remains unclear. In the present study, the potential mechanism of PIG3 participation in the DNA damage response induced by ionizing radiation (IR) was investigated in multiple cell lines with depleted expression of PIG3 transiently or stably by the small interference RNA and lentivirus-mediated shRNA expression strategies. PIG3 knockdown led to an abnormal DNA damage response, including decreased IR-induced phosphorylation of H2AX, Chk1, Chk2 and Kap-1 as well as a prolonged G2-M arrest and aberrant mitotic progression. Notably, PIG3 knockdown resulted in a striking depression of cellular DNA-PKcs protein level, and was accompanied by a downregulation of ATM. Re-expression of PIG3 effectively rescued the depression of DNA-PKcs in PIG3-depleted cells. This negative regulation of DNA-PKcs by depleting PIG3 seemed to take place at the translational level but not at the levels of transcription or protein degradation. However, a compensatory feedback of increased mRNA expression of DNA-PKcs was formed in PIG3-depleted cells after a few passages or cell cycles of subculture, which led the recovery of the DNA-PKcs protein level and the consequent recovered efficiency of the DNA damage response. These results provide a new insight into the mechanism of PIG3s functioning in DNA damage signaling and the regulation network of cellular DNA-PKcs expression homeostasis.

Bystander autophagy mediated by radiation-induced exosomal miR-7-5p in non-targeted human bronchial epithelial cells

Radiation-induced bystander effect (RIBE) describes a set of biological effects in non-targeted cells that receive bystander signals from the irradiated cells. RIBE brings potential hazards to adjacent normal tissues in radiotherapy, and imparts a higher risk than previously thought. Excessive release of some substances from irradiated cells into extracellular microenvironment has a deleterious effect. For example, cytokines and reactive oxygen species have been confirmed to be involved in RIBE process via extracellular medium or gap junctions. However, RIBE-mediating signals and intercellular communication pathways are incompletely characterized. Here, we first identified a set of differentially expressed miRNAs in the exosomes collected from 2 Gy irradiated human bronchial epithelial BEP2D cells, from which miR-7-5p was found to induce autophagy in recipient cells. This exosome-mediated autophagy was significantly attenuated by miR-7-5p inhibitor. Moreover, our data demonstrated that autophagy induced by exosomal miR-7-5p was associated with EGFR/Akt/mTOR signaling pathway. Together, our results support the involvement of secretive exosomes in propagation of RIBE signals to bystander cells. The exosomes-containing miR-7-5p is a crucial mediator of bystander autophagy.

Proteomic profiling revealed the functional networks associated with mitotic catastrophe of HepG2 hepatoma cells induced by 6-bromine-5-hydroxy-4-methoxybenzaldehyde

Mitotic catastrophe, a form of cell death resulting from abnormal mitosis, is a cytotoxic death pathway as well as an appealing mechanistic strategy for the development of anti-cancer drugs. In this study, 6-bromine-5-hydroxy-4-methoxybenzaldehyde was demonstrated to induce DNA double-strand break, multipolar spindles, sustain mitotic arrest and generate multinucleated cells, all of which indicate mitotic catastrophe, in human hepatoma HepG2 cells. We used proteomic profiling to identify the differentially expressed proteins underlying mitotic catastrophe. A total of 137 differentially expressed proteins (76 upregulated and 61 downregulated proteins) were identified. Some of the changed proteins have previously been associated with mitotic catastrophe, such as DNA-PKcs, FoxM1, RCC1, cyclin E, PLK1-pT210, 14-3-3σ and HSP70. Multiple isoforms of 14-3-3, heat-shock proteins and tubulin were upregulated. Analysis of functional significance revealed that the 14-3-3-mediated signaling network was the most significantly enriched for the differentially expressed proteins. The modulated proteins were found to be involved in macromolecule complex assembly, cell death, cell cycle, chromatin remodeling and DNA repair, tubulin and cytoskeletal organization. These findings revealed the overall molecular events and functional signaling networks associated with spindle disruption and mitotic catastrophe.

Proteomic Analysis Implicates Dominant Alterations of RNA Metabolism and the Proteasome Pathway in the Cellular Response to Carbon-Ion Irradiation.

Radiotherapy with heavy ions is considered advantageous compared to irradiation with photons due to the characteristics of the Braggs peak and the high linear energy transfer (LET) value. To understand the mechanisms of cellular responses to different LET values and dosages of heavy ion radiation, we analyzed the proteomic profiles of mouse embryo fibroblast MEF cells exposed to two doses from different LET values of heavy ion 12C. Total proteins were extracted from these cells and examined by Q Exactive with Liquid Chromatography (LC)—Electrospray Ionization (ESI) Tandem MS (MS/MS). Using bioinformatics approaches, differentially expressed proteins with 1.5 or 2.0-fold changes between different dosages of exposure were compared. With the higher the dosage and/or LET of ion irradiation, the worse response the cells were in terms of protein expression. For instance, compared to the control (0 Gy), 771 (20.2%) proteins in cells irradiated at 0.2 Gy of carbon-ion radiation with 12.6 keV/μm, 313 proteins (8.2%) in cells irradiated at 2 Gy of carbon-ion radiation with 12.6 keV/μm, and 243 proteins (6.4%) in cells irradiated at 2 Gy of carbon-ion radiation with 31.5 keV/μm exhibited changes of 1.5-fold or greater. Gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, Munich Information Center for Protein Sequences (MIPS) analysis, and BioCarta analysis all indicated that RNA metabolic processes (RNA splicing, destabilization and deadenylation) and proteasome pathways may play key roles in the cellular response to heavy-ion irradiation. Proteasome pathways ranked highest among all biological processes associated with heavy carbon-ion irradiation. In addition, network analysis revealed that cellular pathways involving proteins such as Col1a1 and Fn1 continued to respond to high dosages of heavy-ion irradiation, suggesting that these pathways still protect cells against damage. However, pathways such as those involving Ikbkg1 responded better at lower dosages than at higher dosages, implying that cell damage would occur when the networks involving these proteins stop responding. Our investigation provides valuable proteomic information for elucidating the mechanism of biological effects induced by carbon ions in general.

DNA-PKcs Negatively Regulates Cyclin B1 Protein Stability through Facilitating Its Ubiquitination Mediated by Cdh1-APC/C Pathway

The catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is a critical component of the non-homologous end-joining pathway of DNA double-stranded break repair. DNA-PKcs has also been shown recently functioning in mitotic regulation. Here, we report that DNA-PKcs negatively regulates the stability of Cyclin B1 protein through facilitating its ubiquitination mediated by Cdh1 / E 3 ubiquitin ligase APC/C pathway. Loss of DNA-PKcs causes abnormal accumulation of Cyclin B1 protein. Cyclin B1 degradation is delayed in DNA-PKcs-deficient cells as result of attenuated ubiquitination. The impact of DNA-PKcs on Cyclin B1 stability relies on its kinase activity. Our study further reveals that DNA-PKcs interacts with APC/C core component APC2 and its co-activator Cdh1. The destruction of Cdh1 is accelerated in the absence of DNA-PKcs. Moreover, overexpression of exogenous Cdh1 can reverse the increase of Cyclin B1 protein in DNA-PKcs-deficient cells. Thus, DNA-PKcs, in addition to its direct role in DNA damage repair, functions in mitotic progression at least partially through regulating the stability of Cyclin B1 protein.

Exosome-packaged miR-1246 contributes to bystander DNA damage by targeting LIG4

BackgroundAn increasing number of studies have recently reported that microRNAs packaged in exosomes contribute to multiple biological processes such as cancer progression; however, little is known about their role in the development of radiation-induced bystander effects.MethodsThe exosomes were isolated from the culture medium of BEP2D cells with or without γ-ray irradiation by ultracentrifugation. To monitor DNA damage and repair efficiency, the DNA double-strand break biomarker 53BP1 foci, comet, micronuclei, expression of DNA repair genes and NHEJ repair activity were detected. The miR-1246 targeting sequence of the DNA ligase 4 (LIG4) mRNA 3′UTR was assessed by luciferase reporter vectors.ResultsmiR-1246 was increased in exosomes secreted from 2 Gy-irradiated BEP2D cells and inhibited the proliferation of nonirradiated cells. The miR-1246 mimic, exosomes from irradiated cells, and radiation-conditioned cell culture medium increased the yields of 53BP1 foci, comet tail and micronuclei in nonirradiated cells, and decreased NHEJ efficiency. miR-1246 downregulated LIG4 expression by directly targeting its 3′UTR.ConclusionsOur findings demonstrate that miR-1246 packaged in exosomes could act as a transfer messenger and contribute to DNA damage by directly repressing the LIG4 gene. Exosomal miR-1246 may be a critical predictor of and player in radiation-induced bystander DNA damage.