Background: When DNA double-strand breaks (DSB) are induced by ionizing radiation (IR) in cells, histone H2AX is quickly phosphorylated into γ-H2AX (p-S139) around the DSB site. The necessity of DNA-PKcs in regulating the phosphorylation of H2AX in response to DNA damage and cell cycle progression was investigated. Results: The level of γH2AX in HeLa cells increased rapidly with a peak level at 0.25 - 1.0 h after 4 Gy γ irradiation. SiRNA-mediated depression of DNA-PKcs resulted in a strikingly decreased level of γH2AX. An increased γH2AX was also induced in the ATM deficient cell line AT5BIVA at 0.5 - 1.0 h after 4 Gy γ rays, and this IR-increased γH2AX in ATM deficient cells was dramatically abolished by the PIKK inhibitor wortmannin and the DNA-PKcs specific inhibitor NU7026. A high level of constitutive expression of γH2AX was observed in another ATM deficient cell line ATS4. The alteration of γH2AX level associated with cell cycle progression was also observed. HeLa cells with siRNA-depressed DNA-PKcs (HeLa-H1) or normal level DNA-PKcs (HeLa-NC) were synchronized at the G1 phase with the thymidine double-blocking method. At ~5 h after the synchronized cells were released from the G1 block, the S phase cells were dominant (80%) for both HeLa-H1 and HeLa-NC cells. At 8 - 9 h after the synchronized cells released from the G1 block, the proportion of G2/M population reached 56 - 60% for HeLa-NC cells, which was higher than that for HeLa H1 cells (33 - 40%). Consistently, the proportion of S phase for HeLa-NC cells decreased to ~15%; while a higher level (26 - 33%) was still maintained for the DNA-PKcs depleted HeLa-H1 cells during this period. In HeLa-NC cells, the γH2AX level increased gradually as the cells were released from the G1 block and entered the G2/M phase. However, this γH2AX alteration associated with cell cycle progressing was remarkably suppressed in the DNA-PKcs depleted HeLa-H1 cells, while wortmannin and NU7026 could also suppress this cell cycle related phosphorylation of H2AX. Furthermore, inhibition of GSK3β activity with LiCl or specific siRNA could up-regulate the γH2AX level and prolong the time of increased γH2AX to 10 h or more after 4 Gy. GSK3β is a negative regulation target of DNA-PKcs/Akt signaling via phosphorylation on Ser9, which leads to its inactivation. Depression of DNA-PKcs in HeLa cells leads to a decreased phosphorylation of Akt on Ser473 and its target GSK3β on Ser9, which, in other words, results in an increased activation of GSK3β. In addition, inhibition of PDK (another up-stream regulator of Akt/GSK3β) by siRNA can also decrease the induction of γH2AX in response to both DNA damage and cell cycle progression. Conclusion: DNA-PKcs plays a dominant role in regulating the phosphorylation of H2AX in response to both DNA damage and cell cycle progression. It can directly phosphorylate H2AX independent of ATM and indirectly modulate the phosphorylation level of γH2AX via the Akt/GSK3 β signal pathway.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is well known as a critical component involving the nonhomologous end joining pathway of DNA double-strand breaks repair. Here, we showed another important role of DNA-PKcs in stabilizing spindle formation and preventing mitotic catastrophe in response to DNA damage. Inactivation of DNA-PKcs by small interfering RNA or specific inhibitor NU7026 resulted in an increased outcome of polyploidy after 2-Gy or 4-Gy irradiation. Simultaneously, a high incidence of multinucleated cells and multipolar spindles was detected in DNA-PKcs-deficient cells. Time-lapse video microscopy revealed that depression of DNA-PKcs results in mitotic catastrophe associated with mitotic progression failure in response to DNA damage. Moreover, DNA-PKcs inhibition led to a prolonged G2-M arrest and increased the outcome of aberrant spindles and mitotic catastrophe in Ataxia-telangiectasia mutated kinase (ATM)–deficient AT5BIVA cells. We have also revealed the localizations of phosphorylated DNA-PKcs/T2609 at the centrosomes, kinetochores, and midbody during mitosis. We have found that the association of DNA-PKcs and checkpoint kinase 2 (Chk2) is driven by Ku70/80 heterodimer. Inactivation of DNA-PKcs strikingly attenuated the ionizing radiation–induced phosphorylation of Chk2/T68 in both ATM-efficient and ATM-deficient cells. Chk2/p-T68 was also shown to localize at the centrosomes and midbody. These results reveal an important role of DNA-PKcs on stabilizing spindle formation and preventing mitotic catastrophe in response to DNA damage and provide another prospect for understanding the mechanism coupling DNA repair and the regulation of mitotic progression. Cancer Res; 70(9); 3657–66. ©2010 AACR.

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.

Increasing evidence indicates that elevated expression of enhancer of zeste homolog 2 gene (EZH2) in many human malignant tumors acts a significant role in the oncogenic process. However, the underlying molecular mechanism is still unclarified. It is evident that apoptosis and autophagy of tumor cells is crucial for the tumorigenesis and progression of cancer, however, the exact role of EZH2 plays in apoptosis and autophagy has not been fully elucidated in colorectal cancer (CRC). Our previous study found that the expression level of EZH2 was higher in CRC tumor tissues than in the paired normal tissues using immunohistochemical analysis. We also recently found that the autophagy‐related gene‐related protein Ambra1 plays an important role in the autophagy pathway in CRC cells. In this study, mRNA and protein expression of EZH2 in four CRC cell lines were tested at first and RKO and HCT116 cells showed the highest levels among them. Here we transfected with EZH2‐shRNA, or added DZNep (an EZH2 inhibitor) to RKO and HCT116 cells in order to detect the effect of EZH2 on autophagy via determining the change of the protein expression of LC3 and Ambra1. The outcome indicated an obvious decrease of autophagy level in cells transfected with EZH2‐shRNA or DZNep. We also found the apoptotic rate of cells was elevated significantly after downregulation of EZH2. In addition, compared to control group, CRC cells transfected with EZH2‐shRNA or added DZNep revealed a significantly increased G1 cell cycle rate and an obvious decrease in the G2 cell cycle rate. Further analysis showed that knockdown of EZH2 induced cell-cycle arrest in CRC cells. Meanwhile, downregulation of EZH2 in CRC cells induces autophagy and apoptosis. Taken together, our results suggest that EZH2 plays a critical role in autophagy and apoptosis in the progression of CRC, which potentially facilitates the development of an ideal strategy for combating colorectal cancer.

Background: C-Myc is a short-lived oncoprotein that is destroyed by ubiquitin-mediated proteolysis. Dysregulated accumulation of c-Myc commonly occurs in human cancers. Some of those cases with the dysregulated c-Myc protein accumulation are attributed to gene amplification or increased mRNA expression. However, the abnormal accumulation of c-Myc protein is also a common finding in human cancers with normal copy number and transcription level of c-Myc gene. It seems that the mechanistic dysregulation in the control of c-Myc protein stabilization is another important hallmark associated with c-Myc accumulation in cancer cells. Here we report a novel mechanistic pathway through which DNA-dependent protein kinase catalytic subunit (DNA-PKcs) modulates the stability of c-Myc protein. Results: Firstly, siRNA-mediated silencing of DNA-PKcs strikingly downregulated c-Myc protein levels in HeLa and HepG2 cells, and simultaneously decreased cell proliferation. The c-Myc protein level in DNA-PKcs deficient human glioma M059J cells was also found much lower than that in DNA-PKcs efficient M059K cells. ATM deficiency does not affect c-Myc expression level. Silencing of DNA-PKcs in HeLa cells resulted in a decreased stability of c-Myc protein, which was associated the increasing of c-Myc phosphorylation on Thr58/Ser62 and ubiquitination level. Phosphorylation of Akt on Ser473, a substrate of DNA-PKcs was found decreased in DNA-PKcs deficient cells. As the consequence, the phosphorylation of GSK3 β on Ser9, a negatively regulated target of Akt, was also decreased, and which led to activation of GSK 3β and in turn phosphorylation of c-Myc on Thr58. Moreover, inhibition of GSK3 activity by LiCl or specific siRNA molecules rescued the downregulation of c-Myc mediated by silencing DNA-PKcs. Consistent with this depressed DNA-PKcs cell model, overexpressing DNA-PKcs in normal human liver L02 cells, by sub-chronically exposing to very low dose of carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), increased c-Myc protein level, the phosphorylation of Akt and GSK3 β, as well as cell proliferation. siRNA-mediated silencing of DNA-PKcs in this cell model reversed above alterations to the original levels of L02 cells. Conclusion: A suitable DNA-PKcs level in cells is necessary for maintaining genomic stability, while abnormal overexpression of DNA-PKcs may contribute to cell proliferation and even oncogenic transformation by stabilizing the c-Myc oncoprotein via at least the Akt/GSK3 pathway. Our results suggest DNA-PKcs a novel biological role beyond its DNA repair function.

The intestine is a highly radiosensitive tissue that is susceptible to structural and functional damage due to systemic as well as localized radiation exposure. Unfortunately, no effective prophylactic or therapeutic agents are available at present to manage radiation-induced intestinal injuries. We observed that the vanillin derivative VND3207 improved the survival of lethally irradiated mice by promoting intestinal regeneration and increasing the number of surviving crypts. Pre-treatment with VND3207 significantly increased the number of Lgr5+ intestinal stem cells (ISCs) and their daughter cells, the transient Ki67+ proliferating cells. Mechanistically, VND3207 decreased oxidative DNA damage and lipid peroxidation and maintained endogenous antioxidant status by increasing the level of superoxide dismutase and total antioxidant capacity. In addition, VND3207 maintained appropriate levels of activated p53 that triggered cell cycle arrest but were not sufficient to induce NOXA-mediated apoptosis, thus ensuring DNA damage repair in the irradiated small intestinal crypt cells. Furthermore, VND3207 treatment restores the intestinal bacterial flora structures altered by TBI exposure. In conclusion, VND3207 promoted intestinal repair following radiation injury by reducing reactive oxygen species-induced DNA damage and modulating appropriate levels of activated p53 in intestinal epithelial cells.