Radiation‐induced DNA damage and repair in peripheral blood mononuclear cells from Nijmegen breakage syndrome patients and carriers assessed by the Comet assay

Abstract

Nijmegen breakage syndrome (NBS) patients and carriers are predisposed to malignancy and are often treated with X-irradiation. In the present study, the single-cell gel electrophoresis (Comet) assay was used to examine radiation-induced DNA damage and repair in peripheral blood mononuclear cells from NBS patients (n=13) and carriers (n=36) of six unrelated families. Cells from apparently healthy donors (n=10) and from breast cancer patients with normal clinical radiosensitivity (n=10) served as controls. Cells were irradiated with 5 Gy of X-rays and assayed for initial DNA damage and for residual DNA damage after 40 min of repair; the kinetics of DNA repair also was estimated. In addition, the nuclear area of unirradiated cells was extracted from the Comet data. The initial radiation-induced DNA fragmentation indicated that cells from members of two out of six NBS families were significantly more sensitive to X-irradiation than cells from the controls. Cells from four NBS families had longer DNA repair half-time values, while cells from five NBS families had significantly increased residual DNA damage following repair. The mean nuclear area of unirradiated cells processed in the Comet assay was 1.3-fold higher in cells from all NBS families than in the controls (P<0.05). Notably, the Comet assay parameters (initial and residual DNA damage and the repair kinetics) of irradiated NBS cells predicted the carrier status of the majority (86%) of blindly tested individuals. The prediction of NBS status was higher if the nuclear area of unirradiated cells was used as the endpoint. The results of this study suggest that the impaired radiation response of NBS cells should be taken into account if radiotherapy of NBS patients and carriers is required.

Intracellular delivery of 2-deoxy-D-glucose into tumor cells by long-term cultivation and through swelling-activated pathways: implications for radiation treatment

“…The Comet assay was performed under alkaline conditions following a protocol reported elsewhere (21,22). DNA fragmentation was quantified from the 'Tail Moment' (TM, given in arbitrary units, a.u.…”

Clinical Applications of the Comet Assay

“…Bu¨rger et al 8 in a study of six Nijmegen breakage syndrome families using peripheral blood mononuclear cells found that two out of the six families were more sensitive to X-irradiation than the controls. DNA repair was also reported to take longer in four out of the six families, while cells from five families with the syndrome had significantly increased residual DNA damage following repair.…”

Nijmegen Breakage Syndrome fibroblasts and iPSCs: cellular models for uncovering disease-associated signaling pathways and establishing a screening platform for anti-oxidants

“…In a study of patients with Cockayne syndrome (CS), a mutation in the repair pathway gene ERCC6 did not impair genetic reprogramming but exhibited elevated cell death rates and ROS production 25 . As NBS cells are hypersensitive to DNA damage 26 , ROS may be detrimental to them under physiological conditions. Thus, it was hypothesized that antioxidants or the induction of pluripotency in NBS fibroblasts might suppress and maybe bypass ROS-mediated genome instability.…”