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Updated: Aug 17, 2026
Assessment of Global DNA Double-Strand End Resection using BrdU-DNA Labeling coupled with Cell Cycle Discrimination Imaging
Published on: April 28, 2021
NBS1 knockdown by small interfering RNA increases ionizing radiation mutagenesis and telomere association in human
Ying Zhang1, Chang U K Lim, Eli S Williams
1Department of Environmental and Radiological Health Sciences, Colorado State University, Fort Collins, Colorado 80523, USA. Ying.Zhang@colostate.edu
Abstract:
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.
Insights
NBS1 gene mutations cause Nijmegen breakage syndrome, increasing cancer risk. NBS1 deficiency in human cells leads to increased mutations and telomere abnormalities, potentially explaining this cancer predisposition.
Area of Science:
- Genetics
- Molecular Biology
- Cancer Research
Background:
- Nijmegen breakage syndrome (NBS) is a rare hereditary disorder caused by hypomorphic mutations in the NBS1 gene, leading to increased cancer predisposition.
- The NBS1 protein is crucial for DNA damage response and maintaining chromosomal integrity as part of the MRE11/RAD50/NBS1 complex.
Purpose of the Study:
- To investigate the cellular phenotypes associated with NBS1 deficiency in human cells.
- To analyze the impact of NBS1 knockdown on DNA damage response, cell cycle checkpoints, and genomic stability in cell lines with different p53 statuses.
Main Methods:
- Utilized small interfering RNA (siRNA) to reduce NBS1 protein levels in TK6 (wild-type p53) and WTK1 (mutated p53) human lymphoblastoid cell lines.
- Assessed ionizing radiation-induced mutations at TK and HPRT loci, phosphorylation of H2AX (gamma-H2AX), Chk2 activation, p53/p21 accumulation, apoptosis, and telomere associations.
Main Results:
- NBS1 knockdown in both cell lines resulted in increased radiation-induced mutations, impaired gamma-H2AX phosphorylation, and reduced Chk2 activation.
- TK6 cells showed decreased radiation-induced apoptosis and p53/p21 accumulation, with differential sensitivity to cell killing compared to WTK1 cells after NBS1 knockdown.
- NBS1 deficiency led to increased telomere associations, independent of radiation exposure and p53 status.
Conclusions:
- NBS1 deficiency in human cells causes hypermutability and telomere associations.
- These observed phenotypes provide experimental evidence linking NBS1 deficiency to the cancer predisposition characteristic of Nijmegen breakage syndrome.
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