No abstract

Human individuals often exhibit important differences in their sensitivity to ionising radiation. Extensive literature links radiation sensitivity with impaired DNA repair which is due to a lack of correct functioning in many proteins involved in DNA-repair pathways and/or in DNA-damage checkpoint responses. Given that ionising radiation is an important and widespread diagnostic and therapeutic tool, it is important to investigate further those factors and mechanisms that underlie individual radiosensitivity. Recently, evidence is accumulating that telomere function may well be involved in cellular and organism responses to ionising radiation, broadening still further the currently complex and challenging scenario.

Combined cytogenetic and biochemical approaches were used to investigate the contributions of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) in the maintenance of genomic stability in nonirradiated and irradiated primary mouse embryo fibroblasts (MEF). We show that telomere dysfunction contributes only marginally to genomic instability associated with DNA-PKcs deficiency in the absence of radiation. Following exposure to ionizing radiation, DNAPKcs À/À MEFs are radiosensitized mainly as a result of the associated DNA double-strand break (DSB) repair defect. This defect manifests as an increase in the fraction of DSB rejoining with slow kinetics although nearly complete rejoining is achieved within 48 hours. Fifty-four hours after ionizing radiation, DNA-PKcs À/À cells present with a high number of simple and complex chromosome rearrangements as well as with unrepaired chromosome breaks. Overall, induction of chromosome aberrations is 6-fold higher in DNA-PKcs À/À MEFs than in their wild-type counterparts. Spectral karyotypingfluorescence in situ hybridization technology distinguishes between rearrangements formed by prereplicative and postreplicative DSB rejoining and identifies sister chromatid fusion as a significant source of genomic instability and radiation sensitivity in DNA-PKcs À/À MEFs. Because DNA-PKcsMEFs show a strong G 1 checkpoint response after ionizing radiation, we propose that the delayed rejoining of DNA DSBs in DNA-PKcs À/À MEFs prolongs the mean life of broken chromosome ends and increases the probability of incorrect joining. The preponderance of sister chromatid fusion as a product of incorrect joining points to a possible defect in S-phase arrest and emphasizes proximity in these misrepair events. (Cancer Res 2005; 65(22): 10223-32)

The disability of cells to rejoin broken chromosome ends does not lead to the healing of DSBs by the acquisition of new telomeric sequences.

Telomeres cap the ends of chromosomes, preventing end-to-end fusions and subsequent chromosome instability. Here we used a telomerase knockout model to investigate whether telomerase participates in the processes of DNA break repair by de novo synthesis of telomere repeats at broken chromosome ends (chromosome healing). Chromosome healing giving rise to new detectable telomeric signals has not been observed in embryonic fibroblasts of telomerase-proficient mice exposed to ionizing radiation. Since the synthesis of telomeric sequences to broken DNA ends would make them refractory to rejoining events, the efficiency of rejoining of broken chromosomes in cell environments with and without telomerase has also been investigated. We conclude that the efficiency of rejoining broken chromosomes is not significantly different in the two cell environments. All together, our results indicate that there is no significant involvement of telomerase in the healing of broken DNA ends by synthesizing new telomeres in mouse embryo fibroblasts after exposure to ionizing radiation.