SPONTANEOUS PREMATURE CHROMOSOME CONDENSATION AND MITOTIC CATASTROPHEFOLLOWING IRRADIATION OF HELA S3 CELLS

Purpose: To study the mechanisms underlying the loss of G(2)/M checkpo int control which leads to mitotic catastrophe in human tumour cells f ollowing exposure to ionizing radiation. Materials and methods: Asynch ronous HeLa S3 cells were irradiated with doses of 5, 10 and 20 Gy X-r ays. Cell-cycle progression and cyclin B1 levels were measured using b ivariate flow-cytometric techniques as a function of time after irradi ation. As indicators of mitotic catastrophe, the appearance of spontan eous premature chromosome condensation (SPCC) and cells presenting nuc lear fragmentation were analysed using microscopy. Cyclin B1-dependent kinase activity was determined in immunoprecipitates and analysed usi ng gel electrophoresis. Results: After X-irradiation of HeLa cells, de lays in late S and G(2) phases of the cell cycle were followed by SPCC and nuclear fragmentation, both indicative of mitotic catastrophe. Th e kinetics of appearance of cells that had apparently undergone mitoti c catastrophe (i.e. the fraction of cells exhibiting nuclear fragmenta tion) was independent of the dose-dependent radiation-induced division delay, while the extent of fragmentation (expressed as the number of nuclear fragments per fragmented cell) did increase with dose. Also ob served was a 5-fold elevation of cyclin BI levels in late S/G(2) cells , which correlated temporally with the observed delays late in the cel l cycle. Following the appearance of elevated cyclin levels, cyclin B1 -associated histone H1 kinase activity showed similar increases; these increases in kinase activity occurred prior to increases in the fract ion of cells exhibiting nuclear fragmentation. Conclusions: In human c ells, cyclin B1 gene expression occurs in late 3 and G(2) phases, and thus the increase observed in this protein may be due to the increased time spent by cells in these phases as a result of cell-cycle delays caused by the radiation exposure. It is possible that, under these con ditions, over accumulation of cyclin B1 dilutes the mitosis-inhibitory action of the weel or other inhibitory pathways. Thus, this study pre sents a possible mechanism for G(2)/M checkpoint abrogation following ionizing radiation which may depend solely on effects associated with perturbed cell-cycle progression.