| DNA-PKcs is the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) complex, in which another two regulatory components are included, i.e, Ku70 and Ku80. DNA-PKcs is also termed PRKDC, and the gene consists of 86 exons distributing in a region of more than 250kb located in chromosome 8. DNA-PKcs has the same concervative domains which are phosphatidylinositol (PI)3-kinase motif, leucine zipper motif, CPP32 incistion site and interaction domain with Ku, c-Abl and KIP proteins in mouse, chicken, Xenopus and human. The whole cDNA of human DNA-PKcs is 13509bp and the open reading frame is 12387bp, encoding a 465-kDa protein with 4127 amino acids. DNA-PKcs is a serine/threonine protein kinase that belongs to the members of the phosphatidylinositol (PI)3-kinase superfamily. This superfamily includes ATM, ATR, RPA and so on. These enzymes are sensitive to wortmannin. In vitro, DNA-PKcs can phosphorylate a number of important proteins including Ku, XRCC4, RPA, c-Abl, p53, C1D, c-myc, c-fos, Top I ,Top II, RNApol I, TF II D, oct-1. DNA-PKcs is a multifunctional protein. It is required for the nonhomolgous end joining (NHEJ) of DNA double-strand breaks and V(D)J recombination. The activity of DNA-PKcs is essential for telomere length maintenance. Because it is a critical kinase involving in the cellular response to environmental genotoxins, we speculated that DNA-PKcs has more function to be explored. So we pay attention to its biological function and mechanisms related to carcinogenesis.To investigate the relationship of function changes of DNA-PKcs with cell carcinogenesis, we have studied the expression and function of the DNA-PKcs in transformed cell lines generated from human bronchial epithelial cells BEP2D by a particles exposure as well as human lung cancer tissues. The main results are as follows:(1) Our results revealed that the capacity of Y -ray-induced DNA DSBs rejoining was markedly decreased in the transformed cell lines of BERP35T1 and BERP35T4derived from a -particle irradiated BEP2D cells. To study its molecule mechanisms, the mRNA expression of seven DNA repair genes (XRCC-1, XRCC-2, XRCC-3, XRCC-4, Ku70, Ku80 and DNA-PKcs) involved in the repair of DNA strand break was analyzed by semiquantitive RT-PCR or Northern blot for each cell line. A 2.5 to 6.5 fold depressing expression of XRCC-2, XRCC-3 and Ku80 (XRCC-5) genes was detected in BERP35T1 cells and BERP35T4 cells as compared with BEP2D cells. The same expression level of XRCC-1, XRCC-4 and Ku70 (XRCC-6) genes was observed among these three cell lines (data not shown). In contrast, BERP35T4 demonstrated about 2.4-fold over expression of the DNA-PKcs gene. Western Blotting was used to estimate the expression of DNA-PKcs and Ku70. Increased expressing level of DNA-PKcs hi BERP35T1 compared with BEP2D cells was also displayed in protein level, but a little decreased expression in BERP35T4. Ku70 expression was the same among three cell lines. Furthermore, we detected DNA-PKcs protein expression level of 14 cases of lung cancer tissues and corresponding marginal normal tissues respectively, the results revealed that DNA-PKcs protein expression level in cancer tissues was much higher than that in corresponding marginal normal tissues. We argued that the expression changes of DNA-PKcs in transformed/cancer cells were dormantly regulated in protein level.(2) In addition, we analyzed the activity of DNA-PKcs by in vitro phosphorylation. A higher level of kinase activity of DNA-PKcs was observed in BERP35T1 as compared with BEP2D. Taken together, we proposed that the expression changes and increased kinase activity of DNA-PKcs could be related to the transformation processing of BEP2D cells initiated by a particles exposure. So, DNA-PKcs is a multifunctional kinase, its over-expression could lead to the activation or inactivation of some tumor-related proteins which result in the disorder of cell proliferation and growth.(3) In order to identify the interacting proteins of DNA-PKcs, its antibody was used to perform im...
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