Temporal Position of G1Arrest in Normal Human Fibroblasts after Exposure to γ-Rays

Abstract G 1 phase cell cycle arrest after exposure to ionizing radiation has been documented in cells with wild-type p53. The temporal location of this arrest within G 1 phase, however, has not been determined. We have now used flow cytometric analysis of bromodeoxyuridine (BrdUrd)-labeled cells to obtain further information about the location of the G 1 phase radiation checkpoint. Human fibroblasts were irradiated with γ-rays and treated with colcemid to stop unlabeled G 2 cells from entering the G 1 phase. Analysis of BrdUrd incorporation revealed that 73% of G 1 phase human lung fibroblasts remain in G 1 phase after exposure to γ-rays, thereby placing the G 1 radiation checkpoint near the end of G 1 phase. The location of the radiation checkpoint correlates with the reported increased expression of cyclin E, increased cyclin E/cdk2 kinase activity, and hyperphosphorylation of pRb in proliferating human fibroblasts.