Radiosensitivity and repair of the inactive X-chromosome. Insights from FISH and immunocytogenetics

The inactive X-chromosome provides a unique opportunity to study the role of transcriptional activity and chromatin condensation in the repair of chromosome damage. We induced chromosome breakage in human lymphocytes with X-rays (1 or 2 Gy) in either G0 and G1 phase of the cell cycle, and in the presence or absence of an inhibitor of double strand break repair, adenine 9-beta-D-arabinofuranoside (Ara-A). Chromosomal aberrations involving the X-chromosome were detected by means of fluorescence in situ hybridization with an X-chromosome specific red painting probe. The activation status of the X-chromosomes involved in the chromosomal aberrations was determined by simultaneous immunocytogenetics with FITC-conjugated antibodies against BrdUrd incorporated at late S-phase to distinguish the late replicating inactive X-chromosome in green-yellow. This multicolor approach allowed us to study and compare breakage and the extent of repair in the active and inactive X-chromosome. Our data indicate that both chromosomes responded with a similar radiosensitivity. This observation was consistent at both X-ray doses and at the two stages of the cell cycle analyzed. However, the number of chromosomal aberrations involving the inactive X-chromosome was increased after repair inhibition with Ara-A. The differential sensitivity to repair inhibition was observed in G0 after 1 Gy and in G1 after 2 Gy. Thus, the activation status of the X-chromosome might be a source of heterogeneity in breakage and repair. These observations suggest that there is heterogeneous repair when the active and the inactive X-chromosomes are compared and that the observed fragility is the result of a compromise between the actual number of breaks induced in each chromosome and their differential processing.