The G₂ Block Induced by DNA Damage: A Caffeine-resistant Component Independent of Cdc25C, MPM-2 Phosphorylation, and H1 Kinase Activity

Abstract

Treatment of cells with agents that cause DNA damage often results in a delay in G₂. There is convincing evidence showing that inhibition of p34^cdc2 kinase activation is involved in the DNA damage-induced G₂ delay. In this study, we have demonstrated the existence of an additional pathway, independent of the p34^cdc2 kinase activation pathway, that leads to a G₂ arrest in etoposide-treated cells. Both the X-ray-induced and the etoposide-induced G₂ arrest were associated with inhibition of the p34^cdc2 H1 kinase activation pathway as judged by p34^cdc2 H1 kinase activity and phosphorylation of cdc25C. Caffeine treatment restored these activities after either of the treatments. However, the etoposide-treated cells did not resume cycling, revealing the presence of an alternative pathway leading to a G₂ arrest. To explore the possibility that this additional pathway involved phosphorylation of the MPM-2 epitope that is shared by a large family of mitotic phosphoproteins, we monitored the phosphorylation status of the MPM-2 epitope after DNA damage and after treatment with caffeine. Phosphorylation of the MPM-2 epitope was depressed in both X-ray and etoposide-treated cells, and the depression was reversed by caffeine in both cases. The results indicate that the pathway affecting MPM-2 epitope phosphorylation is involved in the G₂ delay caused by DNA damage. However, it is not part of the caffeine-insensitive pathway leading to a G₂ block seen in etoposide-treated cells.