Effects of Caffeine on Protein Phosphorylation and Cell Cycle Progression in X-irradiated Two-cell Mouse Embryos

Abstract

The G₂ phase/mitosis transition in cleavage-stage mouse embryos is correlated with an increased phosphorylation of a defined set of proteins at 46, 35, 30, and 29 kDa. Cell cleavage and the associated changes in protein phosphorylation are delayed after X-irradiation. To understand the mechanism of the caffeine-induced uncoupling of mitosis and the cellular reactions to DNA-damaging agents, we have studied the effects of caffeine treatment on cell cycle progression and protein phosphorylation in two-cell mouse embryos after X-irradiation. Caffeine alone had no effect on timing of and changes in phosphorylation associated with the embryonic cell cycle. In combination with X-rays, however, caffeine was able to over-ride the radiation induced G₂ block and restored the normal timing of these phosphorylation changes after X-irradiation. However, new additional changes in protein phosphorylation appeared after the combined treatment. Isobutyl-methylxanthine (IBMX), a substance chemically related to caffeine but a more specific inhibitor of the phosphodiesterase that breaks down cyclic AMP, reduced the radiation induced G₂ block from 4 to 5 h to about 1 h and restored the cell cycle associated changes in protein phosphorylation. However, the same new changes which appeared after the combined treatment of caffeine and X-rays were observed after the combination of IBMX and X-irradiation. IBMX specific changes in protein phosphorylation were detected in both the single and the combined treatment. These results indicate a similar action of caffeine and IBMX in overriding the radiation induced G₂ block in two-cell mouse embryos.