DELAYED DNA-DAMAGE ASSOCIATED WITH MITOTIC CATASTROPHE FOLLOWING X-IRRADIATION OF HELA S3 CELLS

Partial loss of the radiation G2/M, checkpoint is thought to be an ear ly event in cell immortalization, One of the attributes of immortalize d cell lines is an increase in susceptibility to induction of genomic instability by clastogenic agents. Recently we have shown that in irra diated HeLa cells cell cycle delays in late S and G2 lead to overaccum ulation of cyclin B1 and that enhanced intracellular levels of this po sitive regulator of the cell cycle is correlated with cyclin-dependent kinase activation, spontaneous premature chromosome condensation and subsequent mitotic catastrophe occurring following irradiation. Previo us studies have shown that spontaneous premature chromosome condensati on anti mitotic catastrophe are independent of apoptosis, This report shows that 40 h following X-irradiation of HeLa S3 cells, and subseque nt to mitotic catastrophe, DNA strand breaks appear which are chemical ly distinct from those initially produced by ionizing radiation, This delayed damage is recognized by terminal transferase and thus involves generation of free 3'-OH ends. Pulse field gel electrophoresis analys is of DNA size distributions shows that DNA fragments of similar to 40 kbp and smaller are produced. As !strand breaks produced as a direct result of irradiation are generally repaired within a few hours after exposure to X-rays at the doses used, these results describe a novel m echanism for generation of DNA damage occurring a day or more followin g irradiation. These results may be pertinent to the understanding of mechanisms underlying the delayed lethal effects of irradiation and ma y provide an initiating mechanism for radiation-induced genomic instab ility.