The Involvement And Mechanism Of DNA-PKcs In Regulation Of Mitotic Progression

DNA-PKcs is the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) complex, in which there are another two regulatory components, i.e, Ku70 and Ku80. DNA-PKcs is a serine/threonine protein kinase that belongs to the members of the phosphatidyl inositol-3-kinase-like kinase (PIKK) superfamily which includes ATM, ATR and so on. DNA-PKcs was originally recognized to function in the nonhomolgous end joining (NHEJ) pathway of DNA double-strand breaks and V(D)J recombination. In addition, DNA-PKcs was found to be essential for maintaining telomere length and stability, and it can phosphorylate in vitro and in vivo a series of important proteins including some oncogenes products, transduction factors and DNA repair proteins: c-fos, c-myc, oct-1, c-jun, p53, Artimes, XRCC4, H2AX and so on.Recently, overexpression of DNA-PKcs has been reported in a wide variety of human cancers. Our previous work showed that the activity and protein level of DNA-PKcs remarkbly increased in the radiation transformed human branchial epithelial cells. DNA-PKcs could regulate the stability of c-Myc protein through Akt / GSK3β/ c-myc ubquitination signal,pathway, and suggested that the overexpressed activity of DNA-PK may contribute to the metastatic phenotype. The expression level of DNA-PKcs was also reported to correlate with the proliferation activity of some cell types, e.g testis cells in the early stage of spermatogenesis has the highest expression of DNA-PKcs, but the expression significantly decreases after the meiotic division is completed. Some non-productive cells types are defective of DNA-PKcs, suggesting the involvement of DNA-PKcs in cell proliferation. More recently, DNA-PKcs has been shown to function in G2/M checkpoint in response to DNA damage and localize in centrosome. This study is designed to study the involvement and mechanism of DNA-PKcs in the regulation of mitotic progression, especially in response to DNA damage.1) Depletion of DNA-PKcs results in a prolonged G2-M arrest in HeLa cells in response to DNA damage induced 4Gyγ-ray irradiation or the treatment of topoisomerase IIαinhibitor doxorubicin. Simutaneously, a higher percentage of aneuploidy, multinucleated cells (30%-40%) and increased mitotic cells with multipolar spindle were induced in DNA-PKcs silenced HeLa cells than controls. Videomicroscopic observations of the dynamic mitotic progress of DNA-PKcs silenced HeLa cells 24h-48h after 4Gyγ-ray irradiation uncovered a series of abnormal mitotic progression, including delayed metaphase cells, delayed mitotic exit, multi-nucleated cells, and cytokinesis failure. In consequence, the dysreguation of mitotic progression leads to mitosis catastrophe and eventually apoptotic cell death. The same phenomenona were also observed in DNA-PKcs silenced HepG2 cells. 2) The phosphorylated DNA-PKcs at Thr-2609 was found to change its subcellular localization along with the mitotic progression, i.e., it localizes kinetochores and centrosomes in prophase, premetaphase, and metaphase, to sipindle center in anaphase, and to midbody when cell is approaching in the end of cytokinesis. And the phosphorylated DNA-PKcs pT2647 was shown to localize to centrosomes only but not to kinetochores or midbody, indicating that the diversity functions of DNA-PKcs may be phosphorylation site dependent for DNA-PKcs.3) The double immunofluorence staining and confocal observations were performed using antibodies against DNA-PKcs pT2609 and Plk1 antibodies, and demonstrated a colocalization of phospho-DNA-PKcs pT2609 and Plk1 to centrosomes, kinetochores as well as to midbody. We confirmed the interaction of DNA-PKcs with Plk1 in vitro and vive using GST-pull down and co- immunoprecipitation. Western blot analysis of Plk1 protein expression at different time(0h,12h, 24h, 48h) after 4Gyγ-rays irradiation displayed that Plk1 protein can be induced by irradiation, while down -regulated in DNA-PKcs silenced HeLa and HepG2 cells, but the mRNA expression level of Plk1detected by real time PCR shows no difference in both DNA-PKcs deficient and efficient cells. The interaction of DNA-PKcs with Chk2 ws also found in the study. Moreover, IR-induced Chk2 phosphorylation is atttunated by siRNA-mediated depletion of DNA-PKcs and DNA-PKcs inhibitor Nu7026. These results suggested that the regulating effects of DNA-PKcs functions on mitosis may associated with Plk1 and Chk2.4) PIG3 was found to be upregulated by IR in a dose-dependent manner. The interaction of PIG3 with DNA-PKcs, p53 and transcription repressor KAP-1 were unveiled. Moreover, PIG3 could repress p53 transcription activity.5) The interaction and binding domain of c-Myc oncoprotein with DNA- PKcs were determined.The binding site of c-Myc with DNA-PKcs was screened by GST pull-down assay and further comfirmed by the stability detection of mutants via immunofluorescence observation. Binding site of c-Myc was mapped within the domain of 294-370 in its carboxyl terminus. Deletion of this domain was found to increase the stability of c-Myc protein in vivo. c-Myc nagative target p27 and positive target cyclinE were found up-regulated and down-regulated respectively in DNA-PKcs silenced HeLa-H1 cells, suggesting that DNA-PKcs could regulate cell cycle may also though c-Myc protein.Taken together, we have uncovered an important role of DNA-PKcs in regulation of mitotic progression and cell cycle checkpoint in response to DNA damage beyond its DNA repair function. The identification of DNA-PKcs interacting and colocalizing with Plk1 has further provided the mechanistic explanation for this new function of DNA-PKcs. The interactions of DNA-PKcs with c-myc, PIG3 and Kap1 have also been identified. Deficiency of DNA-PKcs reults in dysregulation of mitotic progression at multple acting points upon DNA damage. Our data strongly support the protentiation of DNA-PKcs as anticancer target.