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The G₂ Block Induced by DNA Damage: A Caffeine-resistant Component Independent of Cdc25C, MPM-2 Phosphorylation, and H1 Kinase Activity¹

Departments of Pathology and Laboratory Medicine [R. A. B., R. J. M.] and Radiation Oncology [G. K., W. G. M.], University of Pennsylvania, Philadelphia, Pennsylvania 19104, and Department of Clinical Investigation, University of Texas M. D. Anderson Cancer Center, Houston Texas 77030 [J. K.]

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.

¹ This work was supported by NIH Grants GM47439 (to R. J. M.), GM48457 (to J. K.) and training grant CA01940 (to R. A. B.).

² To whom requests for reprints should be addressed, at Room 269 John Morgan Building, University of Pennsylvania, Philadelphia, PA 19104.

Received 12/29/97. Accepted 4/10/98.

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