Ping-Kun Zhou

Inactivation of DNA-Dependent Protein Kinase Leads to Spindle Disruption and Mitotic Catastrophe with Attenuated Checkpoint Protein 2 Phosphorylation in Response to DNA Damage

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 G(2)-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.

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.

HIV-1 Tat impairs cell cycle control by targeting the Tip60, Plk1 and cyclin B1 ternary complex.

HIV-1 Tat triggers intrinsic and extrinsic apoptosis pathways in both infected and uninfected cells and plays an important role in the pathogenesis of AIDS. Knocking down Tip60, an interactive protein of Tat, leads to the impairment of cell cycle progression, indicating a key role of Tip60 in cell cycle control. We found that Tip60 interacts with Plk1 through its ZnFMYST domain, and that this interaction is enhanced in the G2/M phase. In addition, cyclin B1 was confirmed to interact with the ZnF domain of Tip60. Immunofluorescence imaging showed that Tip60 co-localizes with both Plk1 and cyclin B1 at the centrosome during the mitotic phase and to the mid-body during cytokinesis. Further experiments revealed that Tip60 forms a ternary complex with Plk1 and cyclin B1 and acetylates Plk1 but not cyclin B1. HIV-1 Tat likely forms a quaternary complex with Tip60, cyclin B1 and Plk1. Fluorescent microscopy showed that Tat causes an unscheduled nuclear translocation of both cyclin B1 and Plk1, causing their co-localization with Tip60 in the nucleus. Tat, Tip60, cyclin B1 and Plk1 interactions provide new a mechanistic explanation for Tat-mediated cell cycle dysregulation and apoptosis.

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.

Interaction between HIV-1 Tat and DNA-PKcs modulates HIV transcription and class switch recombination

HIV-1 tat targets a variety of host cell proteins to facilitate viral transcription and disrupts host cellular immunity by inducing lymphocyte apoptosis, but whether it influences humoral immunity remains unclear. Previously, our group demonstrated that tat depresses expression of DNA-PKcs, a critical component of the non-homologous end joining pathway (NHEJ) of DNA double-strand breaks repair, immunoglobulin class switch recombination (CSR) and V(D)J recombination, and sensitizes cells to ionizing radiation. In this study, we demonstrated that HIV-1 Tat down-regulates DNA-PKcs expression by directly binding to the core promoter sequence. In addition, Tat interacts with and activates the kinase activity of DNA-PKcs in a dose-dependent and DNA independent manner. Furthermore, Tat inhibits class switch recombination (CSR) at low concentrations (≤4 µg/ml) and stimulates CSR at high concentrations (≥8 µg/ml). On the other hand, low protein level and high kinase activity of DNA-PKcs promotes HIV-1 transcription, while high protein level and low kinase activity inhibit HIV-1 transcription. Co-immunoprecipitation results revealed that DNA-PKcs forms a large complex comprised of Cyclin T1, CDK9 and Tat via direct interacting with CDK9 and Tat but not Cyclin T1. Taken together, our results provide new clues that Tat regulates host humoral immunity via both transcriptional depression and kinase activation of DNA-PKcs. We also raise the possibility that inhibitors and interventions directed towards DNA-PKcs may inhibit HIV-1 transcription in AIDS patients.

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.

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.

Overexpression of TNKS1BP1 in lung cancers and its involvement in homologous recombination pathway of DNA double‐strand breaks

TNKS1BP1 is a member of the poly(ADP‐ribose) polymerase (PARP) superfamily. Our previous studies have demonstrated that TNKS1BP1 plays an important role in DNA damage response. But whether and how TNKS1BP1 associates with cancer is still not clear. Here, we found that TNKS1BP1 was upregulated in human lung adenocarcinoma (LAC) tissues, and was associated with poor overall survival (OS) in LAC patients. Dysregulation of TNKS1BP1 affected the sensitivity of A549 cells to several DNA damage agents including cisplatin, bleomycin, and ionizing radiation. Mechanically, overexpression of TNKS1BP1 increased the accumulation of S phase cells, which was accompanied by a decrease in M phase cells. More importantly, we found TNKS1BP1 regulated genome stability, mainly through affecting the homologous recombination pathway of DNA double‐strand breaks by inhibiting the RAD51 foci formation. Overall, our study indicates that, in LAC, aberrant expressions of TNKS1BP1 are common events, and overexpression of TNKS1BP1 might affect outcomes of cancer patients to chemotherapy and radiotherapy.