The effects of NBS1 knockdown by small interfering RNA on the ionizing radiation-induced apoptosis in human lymphoblastoid cells with different p53 status.

Mutations of NBS1 are responsible for the human hereditary disease Nijmegen breakage syndrome (NBS), which is characterized by an extremely high cancer rate. In this study, we investigated the influence of NBS1 on ionizing radiation (IR) induced apoptosis. Using small interfering RNA (siRNA) transfection, we knocked down NBS1 protein in three closely related human lymphoblastoid cell lines differing in p53 status: TK6 with a wild-type p53, NH32 with a null mutation of p53, and WTK1 with a mutant p53. We found that up to 48h after 5Gy IR, all three lines showed an obvious induction of apoptosis regardless of the p53 status. The magnitude of apoptosis induction was TK6>NH32>WTK1. This suggested that although p53 is an important modulator of IR-induced apoptosis, other p53-independent apoptosis pathway also exists. Moreover, NBS1 knockdown led to reduction of IR-induced apoptosis in all three lines and both NBS1/ATM/p53/BAX and NBS1/ATM/CHK2/E2F1 apoptosis pathways were partially inactivated. Our results suggest that NBS1 plays an important role in IR-induced apoptosis via both p53-dependent and p53-independent mechanisms. The impaired apoptosis response to DNA damage in NBS1 deficient cells might be one of the important mechanisms of cancer predisposition in NBS patients.

Partial deficiency of DNA-PKcs increases ionizing radiation-induced mutagenesis and telomere instability in human cells.

The correct repair of DNA double-strand breaks (DSBs) is essential to maintaining the integrity of the genome. Misrepair of DSBs is detrimental to cells and organisms, leading to gene mutation, chromosomal aberration, and cancer development. Nonhomologous end-joining (NHEJ) is one of the principal rejoining processes in most higher eukaryotic cells. NHEJ is facilitated by DNA-dependent protein kinase (DNA-PK), which is composed of a catalytic subunit, DNA-PKcs, and the heterodimeric DNA binding regulatory complex Ku70/86. Null mutation of DNA-PKcs leads to immunodeficiency, chromosomal aberration, gene mutation, telomeric end-capping failure, and cancer predisposition in animals and cells. However, it is unknown whether partial deficiency of DNA-PKcs as might occur in a fraction of the population (e.g., heterozygotes), influences cellular function. Using small interfering RNA (siRNA) transfection, we established partial deficiency of DNA-PKcs in human cells, ranging from 4 to 85% of control levels. Our results reveal for the first time, that partial deficiency of DNA-PKcs leads to increased ionizing radiation (IR)-induced mutagenesis, cell killing, and telomere dysfunction. Radiation mutagenesis was increased inversely with DNA-PKcs protein level, with the most pronounced effect being observed in cells with protein levels below 50% of controls. A small but statistically significant increase in IR-induced cell killing was observed as DNA-PKcs levels decreased, over the entire range of protein levels. Frequencies of IR-induced telomere-DSB fusion was increased at levels of DNA-PKcs as low as approximately 50%, similar to what would be expected in heterozygous individuals. Taken together, our results suggest that even partial deficiency of DNA repair proteins may represent a considerable risk to genomic stability.

NBS1 knockdown by small interfering RNA increases ionizing radiation mutagenesis and telomere association in human cells.

Hypomorphic mutations which lead to decreased function of the NBS1 gene are responsible for Nijmegen breakage syndrome, a rare autosomal recessive hereditary disorder that imparts an increased predisposition to development of malignancy. The NBS1 protein is a component of the MRE11/RAD50/NBS1 complex that plays a critical role in cellular responses to DNA damage and the maintenance of chromosomal integrity. Using small interfering RNA transfection, we have knocked down NBS1 protein levels and analyzed relevant phenotypes in two closely related human lymphoblastoid cell lines with different p53 status, namely wild-type TK6 and mutated WTK1. Both TK6 and WTK1 cells showed an increased level of ionizing radiation-induced mutation at the TK and HPRT loci, impaired phosphorylation of H2AX (gamma-H2AX), and impaired activation of the cell cycle checkpoint regulating kinase, Chk2. In TK6 cells, ionizing radiation-induced accumulation of p53/p21 and apoptosis were reduced. There was a differential response to ionizing radiation-induced cell killing between TK6 and WTK1 cells after NBS1 knockdown; TK6 cells were more resistant to killing, whereas WTK1 cells were more sensitive. NBS1 deficiency also resulted in a significant increase in telomere association that was independent of radiation exposure and p53 status. Our results provide the first experimental evidence that NBS1 deficiency in human cells leads to hypermutability and telomere associations, phenotypes that may contribute to the cancer predisposition seen among patients with this disease.