Spontaneous premature chromosome condensation and mitotic catastrophe following irradiation of HeLa S3 cells

Abstract

Purpose : To study the mechanisms underlying the loss of G2 /M checkpoint control which leads to mitotic catastrophe in human tumour cells following exposure to ionizing radiation. Materials and methods : Asynchronous HeLa S3 cells were irradiated with doses of 5, 10 and 20 Gy X-rays. Cell-cycle progression and cyclin B1 levels were measured using bivariate flow-cytometric techniques as a function of time after irradiation. As indicators of mitotic catastrophe, the appearance of spontaneous premature chromosome condensation (SPCC) and cells presenting nuclear fragmentation were analysed using microscopy. Cyclin B1- dependent kinase activity was determined in immunoprecipitates and analysed using gel electrophoresis. Results : After X-irradiation of HeLa cells, delays in late S and G2 phases of the cell cycle were followed by SPCC and nuclear fragmentation, both indicative of mitotic catastrophe. The kinetics of appearance of cells that had apparently undergone mitotic catastrophe (i.e. the fraction of cells exhibiting nuclear fragmentation) 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 observed was a 5-fold elevation of cyclin B1 levels in late S/G2 cells, which correlated temporally with the observed delays late in the cell 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 fraction of cells exhibiting nuclear fragmentation. Conclusions : In human cells, cyclin B1 gene expression occurs in late S and G2 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 conditions, over accumulation of cyclin B1 dilutes the mitosis-inhibitory action of the wee1 or other inhibitory pathways. Thus, this study presents a possible mechanism for G2 /M checkpoint abrogation following ionizing radiation which may depend solely on effects associated with perturbed cell-cycle progression.