Evaluation of sub-G1 peak in mitotic catastrophe

Abstract

Flow cytometric techniques are used widely in studies of cell death, and particularly in the identification of apoptotic cells. Darzynkiewicz reviewed methods of detecting apoptosis by the analysis of DNA fragmentation. Mitotic catastrophe is an important mechanism of inducing cell death in cancer cells by radiation and hyperthermia, which damage DNA. This process is facilitated by defects in the G1 and G2 checkpoints of the cell cycle, which allow the cells to enter mitosis with DNA damage. DNA fragmentation and micronuclei have also been observed in mitotic catastrophe, and a sub-G1 peak is therefore detected. To elucidate the effects of radiation on cell death, we examined the cell cycle in KK47 bladder cancer cells following irradiation. Twenty-four hours after 5-Gy irradiation, irradiated cells were arrested in G2 phase. Laser scanning cytometry performed 48 h after irradiation revealed the following two pathways : 1) catastrophic cell death ; or 2) failure to undergo cytokinesis, resulting in polyploidy. Nuclear fragmentation and micronuclei were observed in the accumulating sub-G1 population 48 h after irradiation. The tumor suppressor protein p53 is a critical regulator of the G1/S and G2 checkpoints. We found that short interfering RNA-mediated silencing of p53 in normal human fibroblasts, together with DNA damage by irradia tion, efficiently induced mitotic catastrophe. In this experiment, too, nuclear fragmentation and micronuclei were observed in the accumulating sub-G1 population. In conclusion, when a sub-G1 peak is observed by cytometry, not only apoptosis, but also mitotic cell death,may be present.