In normal human fibroblasts variation in DSB repair capacity cannot be ascribed to radiation-induced changes in the localisation, expression or activity of major NHEJ proteins

Abstract 

Background and purpose

The aim of the present study was to test whether for normal human fibroblasts the variation in double-strand break (DSB) repair capacity results from radiation-induced differences in localisation, expression or activity of major non-homologous end-joining (NHEJ) proteins.

Materials and methods

Experiments were performed with 11 normal human fibroblast strains AF01-11. NHEJ proteins were determined by Western blot and DNA-PK activity by pulldown-assay.

Results

The four NHEJ proteins tested (Ku70, Ku80, XRCC4 and DNA-PKcs) were found to be localised almost exclusively in the nucleus with no detectable amount in the cytoplasm. This distribution was not altered upon irradiation. In non-irradiated cells the level of these proteins varied with a CV ranging between 16% and 20%, but there was no correlation with the respective cellular DSB repair capacity. Irradiation (3.5 and 15Gy) did not alter the expression of these proteins and there was also no change in the DNA-PK activity. These results indicate that the variation in DSB repair capacity determined for these fibroblasts can be ascribed to differences neither in the localisation or expression of Ku70, Ku80 and XRCC4 nor in the activity of the DNA-PK complex induced upon irradiation.

Conclusions

For normal human fibroblasts, the level or activity of NHEJ proteins measured prior to or after irradiation cannot be used to predict the DSB repair capacity or cellular radiosensitivity.

Abbreviations: BCA, bicinchoninic acid, cpm, counts per minute, CV, coefficient of variation, DSB, double-strand break, ECL, enhanced chemiluminescence, HDR, homology-directed repair, NHEJ, non-homologous end-joining, PCNA, proliferating cell nuclear antigen, PV, pellet volume, PVDF, polyvinylidene difluoride, SNP, single nucleotide polymorphism, WCE, whole cell extract

Keywords: Repair protein, Ku70/80, XRCC4, DNA-PK activity, Ionising radiation, Induction