G₂/M is a critical phase to regulate X-ray-induced apoptosis in EL-4 mouse lymphoma cells

Abstract

The relationship between apoptosis induction by X-ray and by cell cycle regulation remains not well understood. In this study, using EL-4 mouse lymphoma cells we demonstrate that the majority of apoptotic cell death following X-irradiation is induced after G₂/M arrest. When cells were irradiated at a dose of 20Gy, their viability began to decrease at 24h. Gel electrophoresis of the extracted DNA showed multimers of 200bp fragments, which are characteristic of apoptosis. An increase in the apoptotic fraction was observed when the X-irradiated cells began to accumulate in the G₂/M phase. The apoptotic rate was remarkably augmented by caffeine that inhibits the G₂ checkpoint system; this effect was clearly demonstrated by a suppression of the cyclin B1: cdc2 ratio. A remarkable decrease in bcl-2 expression may also be ascribed to the apoptosis-enhancing effect by caffeine. In contrast, an antimitotic drug, colchicine, did not affect the rate of DNA fragmentation. These results suggest that G₂/M is a critical phase in the regulation of apoptotic cell death and that X-ray-induced apoptosis can be triggered if mitotic events progress without repair of the damaged DNA. Despite X-irradiation's usual effect in increasing p53 expression, intracellular p53 levels in irradiated cells were significantly suppressed in the presence of caffeine. This demonstrates that the mechanism of X-ray-induced apoptosis is involved in the increased expression of p53 proteins, whereas enhancement of apoptotic cell death by caffeine is independent of p53 status.