Genomic Maintenance and Repair

Sunday, October 16, 2005 1:30 PM-3:00 PM Room No. 603
Chair(s): Chen, David; Powell, Simon

(MS004) NBS1 knockdown increases telomere association and radiation mutagenesis in human cells.

Zhang, Ying*,1, Lim, Chang1, Williams, Eli1, Zhou, Junqing1, Zhang, Qinming1, Fox, Michael1, Bailey, Susan 1, Liber, Howard1, 1 Department of Environmental and Radiological Science, Ft. Collins, CO, USA

ABSTRACT- The NBS1 protein is a component of the MRE11/RAD50/NBS1 (MRN) complex that plays a critical role in cellular responses to DNA damage and the maintenance of chromosomal integrity. Inactivation of the NBS1 gene is responsible for Nijmegen breakage syndrome (NBS), a rare autosomal recessive hereditary disorder that imparts an increased predisposition to development of malignancy. Although the molecular mechanism of this cancer predisposition is still unknown, the phenotypic characteristics of NBS1 deficiency in human cells suggest that compromised DNA double strand break (DSB) repair capability may be responsible. If so, this ought also to lead to increased mutagenesis, direct evidence for this hypothesis is still lacking. Utilizing small interfering RNA (siRNA) 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 (IR)-induced gene mutation at the TK and HPRT loci, impaired phosphorylation of H2AX, and impaired activation of the cell cycle checkpoint regulator Chk2. In TK6 cells, IR-induced activation of p53/p21 and apoptosis were reduced. Interestingly, there was a differential response to IR-induced cell killing between TK6 and WTK1 cells after NBS1 knockdown; TK6 cells were more resistant to killing while WTK1 cells were more sensitive. NBS1 deficiency also resulted in a significant increase in telomere association (TA) 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 TA, phenotypes that may contribute to the cancer predisposition seen among patients with this disease.

Key words: NBS1, Radiation, Telomere association, Mutagenesis

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2005 RRS