The Study On The Regulation Of DNA-PKcs To C-myc Stability

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 phosphatidylinositol(PI)3-kinase-related protein kinase (PIKK) superfamily which includes ATM, ATR and so on. DNA-PKcs was originally recognized to function in the pathways of the nonhomolgous end joining (NHEJ) of DNA double-strand breaks and V(D)J recombination. In addition, DNA-PKcs is essential for maintaining telomere length and stability, and it can phosphorylate in vitro a number of important proteins including some oncogenes products and transduction factors: c-fos, c-myc, oct-1, c-jun and so on.Recently, overexpression of DNA-PKcs has been reported in a wide variety of human tumors. Hosoi et al reported that the activity and protein/mRNA levels of DNA-PKcs were significantly higher in the tumor tissues as compared with the normal tissues. Um et al have revealed the increase protein level and activity of DNA-PKcs in the metastatic cancer cell lines as compared with their parental cells, and suggested that the activities of DNA-PK may contribute to the metastatic phenotype. Moveover, the overexpression of DNA-PKcs was also found in brain tumors and liver neoplasm etc. The expression level of DNA-PKcs was also reported to correlate with the development of tissues or the differentiation status of some cell types, e.g testis cells in the early stages of spermatogenesis were the highest expression of DNA-PKcs, but the expression is down-regulated after both meiotic divisions are completed. Some non-productive cells types were negative for DNA-PKcs. Our previous work showed that the activity and protein level of DNA-PKcs in the radiation transformed cells obviously increased. The result of immunohistochemistry indicated that the DNA-PKcs level of liver cancer was higher than that of adjacent normal cells. The activity and protein of DNA-PKcs in lung cancers were also higher as compared with the adjacent normal cells. Based on ourprevious works, this study is designed to reveal more function of DNA-PKcs and to explore the potential of DNA-PKcs as an anticancer target.1) In this section, HeLa cells were treated with DNA-PKcs inhibitor TFP and Y -irradiation. The results showed that TFP could significantly inhibit the growth of HeLa cells and induce apoptosis in a concentration- and time-dependent manner. TFP as well as y-irradiation could induce the degradation of DNA-PKcs, which was considered to facilitate the apoptosis and cellular radiosensitivity. It was proposed that the activity of MAPK p38 in HeLa cells plays a negative control role in apoptosis induction, and inhibiting the phosphorylation of p38 could efficiently prompt cell apoptosis. Our results indicated that TFP could inhibit the phosphorylation of p38 induced by ionizing radiation.2) In order to further investigate the function of DNA-PKcs in rumor occurance and malignant transformation, RNA interference technique was used to silence the expression of DNA-PKcs. Stable transfectants were selected from the HeLa cells transfected with DNA-PKcs siRNA-expressing vector. The expression of DNA-PKcs was significantly suppressed by siRNA. The sensitivity of these DNA-PKcs knock down cells to ionizing and UV irradiation was proved increased. The growth of cultured cells as well as graft tumor cells in BALB/c nude mice was suppressed in certain content, and these results implied a link between DNA-PKcs and tumor cells growth.3) Microarray assay was used to explore the transcriptional profiling of signal transduction-associated genes. The results demonstrated that 15 genes were up-regulated and 8 were down-regulated in DNA-PKcs knock down cells compared with the control cells. Half of the up-regulated genes are associated with the interferon signaling events. The down-regulated genes include some regulators of cell proliferation and differentiation. The semi-quantitative RT-PCR proved the results of microarray assay. The SEAP reporter expression system further confirmed that the transaction activity of the down-regulated NFAT was markedly minimized. These results demonstrated that DNA-PKcs could alter the transcriptional level of certain signal transduction-associated genes related to proliferation and differentiation. Some of them are target genes of c-myc protein such asp21,p27andM)/?G7.4) The potential effect of DNA-PKcs on c-myc was further investigated. Western blotting assay showed that the protein expression of oncogene c-myc obviously suppressed and its transaction activity on target gene in the cells was also inhibited. DNA-PKcs inhibitor wortmannin had the same supprssed effect on the c-myc protein expression and transaction activity. While the mRNA level of c-myc almost had no change under the same condition. Immunoprecipitation assay demonstrated the interaction between DNA-PKcs protein and c-myc protein. These results demonstrated that DNA-PKcs could play a role in maintaining the stability of c-myc protein and DNA-PKcs could involve in the process of the cellular malignant transformation through the pathway regulating c-myc.5) It is known that the stability of c-myc protein is mainly regulated by the ubiquitinization-related proteasome mediated degradation. Based on the latest literature and above date we raise a process of DNA-PKcs/Akt/GSK3/c-myc signal pathway for regulating c-myc stability. In order to confirm this pathway, DNA-PKcs knock down cells and control cells were treated with proteasesome inhibitor MG132 and GSK3 inhibitor LiCl to investigate whether these treatments could reverse the suppressing effect of silencing DNA-PKcs on c-myc stability. After these managements, c-myc expression in the DNA-PKcs knock down cells was recovered. This information provides a primary key data to surpport the DNA-PKcs/Akt /GSK3/c-myc signal passway. But more investigation should be done to validate this passway.6) Our previous work displayed that DNA-PKcs can interact with CyclinT2. CyclinT2 is one of important proteins involving in the process of gene transcription. So we presumed that DNA-PKcs could play a role in DNA transcription-coupled repair (TCR). In order to validate this hypothsis, we firstly established a new technique detecting DNA TCR based on DNA strand-specific PCR. By using this method, repair of UV-induced damage in DHFR gene transcribe- and nontranscribe-strand was compared between DNA-PKcs knock down cells and control cells. The results showed that the repair of transcribe strand in control cells is quicker than the nontranscribe strand and is also remarkedly quicker than...